Modular assembly of antibody genes, antibodies prepared thereby and use

ABSTRACT

The invention relates to cDNA genetic sequences, vehicles containing same as well as hosts transformed therewith, for the production of chimeric immunoglobulin molecules, functional fragments thereof and immunoglobulin derivatives exhibiting novel functional properties comprising human constant region modules and non-human variable region modules, or for class switching antibody molecules and/or chains. 
     The invention also relates to DNA coding for pectate lyase signal peptide has been cloned on a plasmid to create a secretion vector which is capable of producing a chosen protein which is transported across the bacterial membrane. The secretion vector has been used to secrete extracellular thaumatin and extracellular chimeric antibody fragments. The proteins produced by this vector have biological activity. The thaumatin is properly folded and the antibody fragments are capable-of binding antigens on target cancer cells. The invention also relates to the secretion of chimeric antibodies and fragments thereof from yeast in functional form.

This application is a continuation of U.S. application Ser. No.07/987,555, filed Dec. 8, 1992 (abandoned) which is a continuation ofU.S. application Ser. No. 07/501,092, filed Mar. 29, 1990 (abandoned),which is a continuation-in-part of U.S. application Ser. No. 07/077,528,filed Jul. 24, 1987 (abandoned), which is a continuation-in-part ofPCT/US86/02269, filed Oct. 27, 1986 (abandoned), which is acontinuation-in-part of U.S. application Ser. No. 06/793,980, filed Nov.1, 1985 (abandoned); U.S. application Ser. No. 07/501,092, filed Mar.29, 1990 is also a continuation-in-part of U.S. application Ser. No.07/142,039, filed Jan. 11, 1988 (abandoned).

FIELD OF THE INVENTION

This invention relates to recombinant DNA methods of preparingimmunoglobulins, genetic sequences coding therefor, as well as methodsof obtaining such sequences.

BACKGROUND ART

The application of cell-to-cell fusion for the production of monoclonalantibodies by Kohler and Milstein (Nature (London), 256: 495, 1975) hasspawned a revolution in biology equal in impact to the invention ofrecombinant DNA cloning. Hybridoma-produced monoclonal antibodies arealready widely used in clinical diagnoses and basic scientific studies.Applications of human B cell hybridoma-produced monoclonal antibodieshold great promise for the clinical treatment of cancer, viral andmicrobial infections, B cell immunodeficiencies with diminished antibodyproduction, and other diseases and disorders of the immune system.

Unfortunately, yields of monoclonal antibodies from human hybridoma celllines are relatively low (1 ug/ml In human×human compared to 100 ug/mlIn mouse hybridomas), and production costs are high for antibodies madein large scale human tissue culture. Mouse×mouse hybridomas, on theother hand, are useful because they produce abundant amounts of protein,and these cell lines are more stable than the human lines. However,repeated injections of "foreign" antibodies, such as a mouse antibody,in humans, can lead to harmful hypersensitivity reactions.

There has therefore been recent exploration of the possibility ofproducing antibodies having the advantages of monoclonals frommouse-mouse hybridomas, yet the species specific properties of humanmonoclonal antibodies.

Another problem faced by immunologists is that most human monoclonalantibodies (i.e., antibodies having human recognition properties)obtained in cell culture are of the IgM type. When it is desirable toobtain human monoclonals of the IgG type, however, it has been necessaryto use such techniques as cell sorting, to separate the few cells whichhave switched to producing antibodies of the IgG or other type from themajority producing antibodies of the IgM type. A need therefore existsfor a more ready method of switching antibody classes, for any givenantibody of a predetermined or desired antigenic specificity.

The present Invention bridges both the hybridoma and monoclonal antibodytechnologies and provides a quick and efficient method, as well asproducts derived therefrom, for the improved production of chimerichuman/non-human antibodies, or of "class switched" antibodies.

INFORMATION DISCLOSURE STATEMENT*

Approaches to the problem of producing chimeric antibodies have beenpublished by various authors.

Morrison, S. L. et al., Proc. Natl. Acad. Sci., USA, 81:6851-6855(November 1984), describe the production of a mouse-human antibodymolecule of defined antigen binding specificity, produced by joining thevariable region genes of a mouse antibody-producing myeloma cell linewith known antigen binding specificity to human immunoglobulin constantregion genes using recombinant DNA techniques. Chimeric genes wereconstructed, wherein the heavy chain variable region exon from themyeloma cell line S107 well joined to human IgG1 or IgG2 heavy chainconstant region exons, and the light chain variable region exon from thesame myeloma to the human kappa light chain exon. These genes weretransfected into mouse myeloma cell lines and transformed cellsproducing chimeric mouse-human antiphosphocholine antibodies were thusdeveloped.

Morrison, S. L. et al., European Patent Publication No. 173494(published Mar. 5, 1986), disclose chimeric "receptors" (e.g.antibodies) having variable regions derived from one species andconstant regions derived from another. Mention is made of utilizing cDNAcloning to construct the genes, although no details of cDNA cloning orpriming are shown. (see pp 5, 7 and 8).

Boulianne, G. L. et al., Nature, 312:643 (Dec. 13, 1984), also producedantibodies consisting of mouse variable regions joined to human constantregions. They constructed immunoglobulin genes in which the DNA segmentsencoding mouse variable regions specific for the hapten trinitrophenyl(TNP) were joined to segments encoding human mu and kappa constantregions. These chimeric genes were expressed as functional TNP bindingchimeric IgM.

For a commentary on the work of Boulianne et al. and Morrison et al.,see Munro, Nature, 12: 597 (Dec. 13, 1984), Dickson, Genetic EngineeringNews, 5, No. 3 (March 1985), or Marx, Science, 229: 455 (August 1985).

Neuberger, M. S. et al., Nature, 314:268 (Mar. 25, 1986), alsoconstructed a chimeric heavy chain immunoglobulin gene in which a DNAsegment encoding a mouse variable region specific for the hapten4-hydroxy-3-nitrophenacetyl (NP) was Joined to a segment encoding thehuman epsilon region. When this chimeric gene was transfected into theJ558L cell line, an antibody was produced which bound to the NP haptenand had human IgE properties.

Neuberger, M. S. et al., have also published work showing thepreparation of cell lines that secrete hapten-specific antibodies inwhich the Fc portion has been replaced either with an active enzymemoiety (Williams, G. and Neuberger, M. S. Gene 43:319, 1986) or with apolypeptide displaying c-myc antigenic determinants (Nature, 312:604,1984).

Neuberger, M. et al., PCT Publication WO 86/01533, (published Mar. 13,1986) also disclose production of chimeric antibodies (see p. 5) andsuggests, among the technique's many uses the concept of "classswitching" (see p. 6).

Taniguchi, M., in European Patent Publication No. 171 496 (publishedFeb. 19, 1985) discloses the production of chimeric antibodies havingvariable regions with tumor specificity derived from experimentalanimals, and constant regions derived from human. The correspondingheavy and light chain genes are produced in the genomic form, andexpressed in mammalian cells.

Takeda, S. et al., Nature, 314: 452 (Apr. 4, 1985) have described apotential method for the construction of chimeric immunoglobulin geneswhich have intron sequences removed by the use of a retrovirus vector.However, an unexpected splice donor site caused the deletion of the Vregion leader sequence. Thus, this approach did not yield completechimeric antibody molecules.

Cabilly, S. et al., Proc. Natl. Acad. Sci., USA, 81:3273-3277 (June1984), describe plasmids that direct the synthesis in E. coli of heavychains and/or light chains of anti-carcinoembryonic antigen (CEA)antibody. Another plasmid was constructed for expression of a truncatedform of heavy chain (Fd') fragment in E. coli. Functional CEA-bindingactivity was obtained by in vitro reconstitution, in E. coli extracts,of a portion of the heavy chain with light chain.

Cabilly, S., et al., European Patent Publication 25023 (published Nov.14, 1984) describes chimeric immunoglobulin genes and their presumptiveproducts as well as other modified forms. On pages 21, 28 and 33 itdiscusses cDNA cloning and priming.

Boss, M. A., European Patent Application 120694 (published Oct. 3, 1984)shows expression in E. coli of non-chimeric immunoglobulin chains with4-nitrophenyl specificity. There is a broad description of chimericantibodies but no details (see p. 9).

Wood, C. R. et al., Nature, 314:446 (April, 1985) describe plasmids thatdirect the synthesis of mouse anti-NP antibody proteins in yeast. Heavychain mu antibody proteins appeared to be glycosylated in the yeastcells. When both heavy and light chains were synthesized in the samecell, some of the protein was assembled into functional antibodymolecules, as detected by anti-NP binding activity in soluble proteinprepared from yeast cells.

Alexander, A. et al., Proc. Nat. Acad. Sci. USA, 79: 3260-3264 (1982),describe the preparation of a cDNA sequence coding for an abnormallyshort human Ig gamma heavy chain (OMM gamma³ HCD serum protein)containing a 19- amino acid leader followed by the first 15 residues ofthe V region. An extensive internal deletion removes the remainder ofthe V and the entire C_(H) 1 domain. This is cDNA coding for aninternally deleted molecule.

Dolby, T. W. et al., Proc. Natl. Acad. Sci., USA, 77: 6027-6031 (1980),describe the preparation of a cDNA sequence and recombinant plasmidscontaining the same coding for mu and kappa human immunoglobulinpolypeptides. One of the recombinant DNA molecules contained codons forpart of the C_(H) 3 constant region domain and the entire 3' noncodingsequence.

Seno, M. et al., Nucleic Acids Research, 11: 719-726 (1983), describethe preparation of a cDNA sequence and recombinant plasmids containingthe same coding for part of the variable region and all of the constantregion of the human IgE heavy chain (epsilon chain).

Kurokawa, T. et al., ibid, 11: 3077-3085 (1983), show the construction,using cDNA, of three expression plasmids coding for the constant portionof the human IgE heavy chain.

Liu, F. T. et al., Proc. Nat. Acad. Sci., USA, 81: 5369-5373 (September1984), describe the preparation of a cDNA sequence and recombinantplasmids containing the same encoding about two-thirds of the C_(H) 2,and all of the C_(H) 3 and C_(H) 4 domains of human IgE heavy chain.

Tsujimoto, Y. et al., Nucleic Acids Res., 12: 8407-8414 (November 1984),describe the preparation of a human V lambda cDNA sequence from an Iglambda-producing human Burkitt lymphoma cell line, by taking advantageof a cloned constant region gene as a primer for cDNA synthesis.

Murphy, J., PCT Publication WO 83/03971 (published Nov. 24, 1983)discloses hybrid proteins made of fragments comprising a toxin and acell-specific ligand (which is suggested as possibly being an antibody).

Tan, et al., J. Immunol. 135:8564 (November, 1985), obtained expressionof a chimeric human-mouse immunoglobulin genomic gene after transfectioninto mouse myeloma cells.

Jones, P. T., et al., Nature 321:552 (May 1986) constructed andexpressed a genomic construct where CDR domains of variable regions froma mouse monoclonal antibody were used to substitute for thecorresponding domains in a human antibody.

Sun, L. K., et al., Hybridoma 5 suppl. 1 S17 (1986), describes achimeric human/mouse antibody with potential tumor specificity. Thechimeric heavy and light chain genes are genomic constructs andexpressed in mammalian cells.

Sahagan et al., J. Immun. 37:1066-1074 (August 1986) describe a chimericantibody with specificity to a human tumor associated antigen, the genesfor which are assembled from genomic sequences.

For a recent review of the field see also Morrison, S. L., Science229:1202-1207 (Sep. 20, 1985) and Oi, V. T., et al., BioTechniques 4:214(1986).

The Oi, et al., paper is relevant as it argues that the production ofchimeric antibodies from cDNA constructs in yeast and/or bacteria is notnecessarily advantageous.

See also Commentary on page 835 in Biotechnology 4 (1986).

SUMMARY OF THE INVENTION

The invention provides a novel approach for producing geneticallyengineered antibodies of desired variable region specificity andconstant region properties through gene cloning and expression of lightand heavy chains. The cloned immunoglobulin gene products can beproduced by expression in genetically engineered organisms.

The application of chemical gene synthesis, recombinant DNA cloning, andproduction of specific immunoglobulin chains in various organismsprovides an effective solution for the efficient large scale productionof human monoclonal antibodies. The invention also provides a solutionto the problem of class switching antibody molecules, so as to readilyprepare immunoglobulins of a certain binding specificity of any givenclass.

The invention provides cDNA sequences coding for immunoglobulin chainscomprising a constant human region and a variable, either human ornon-human, region. The immunoglobulin chains can either be heavy orlight.

The invention also provides gene sequences coding for immunoglobulinchains comprising a cDNA variable region of either human or non-humanorigin and a genomic constant region of human origin.

The invention also provides genes sequences coding for immunoglobulinchains with secretion signal sequences of prokaryotic or eukaryoticorigin.

The invention also provides sequences as above, present in recombinantDNA molecules, especially in vehicles such as plasmid vectors, capableof expression in desired prokaryotic or eukaryotic hosts.

The invention also provides a gene sequence having a single bacterialpromoter coding a dicistronic message for the expression of multipleheavy and light chains.

The invention also provides consensus sequences and specificoligonucleotide sequences useful as probes for hybridization and primingcDNA synthesis of any hybridoma mRNA coding for variable regions of anydesired specificity.

The invention provides hosts capable of producing, by culture, chimericantibodies and methods of using these hosts.

The invention also provides chimeric immunoglobulin individual chains,whole assembled molecules, and immunoglobulin fragments (such as Fab)having human constant regions and non-human variable regions, whereinboth variable regions have the same binding specificity.

Among other immunoglobulin chains and/or molecules provided by theinvention are:

(a) a complete functional, immunoglobulin molecule comprising:

(i) two identical chimeric heavy chains comprising a non-human variableregion and human constant region and

(ii) two identical all (i.e. non-chimeric) human light chains.

(b) a complete, functional, immunoglobulin molecule comprising:

(i) two identical chimeric heavy chains comprising a non-human variableregion and a human constant region, and

(ii) two identical all (i.e. non-chimeric) non-human light chains.

(c) a monovalent antibody, i.e., a complete, functional immunoglobulinmolecule comprising:

(i) two identical chimeric heavy chains comprising a non-human variableregion and a human constant region, and

(ii) two different light chains, only one of which has the samespecificity as the variable region of the heavy chains.

The resulting antibody molecule binds only to one end thereof and isthere-fore fore incapable of divalent binding;

(d) an antibody with two different specificities, i.e., a complete,functional immunoglobulin molecule comprising:

(i) two different chimeric heavy chains, the first one of whichcomprises a non-human variable region and a human constant region andthe second comprises a different non-human variable region, and a humanconstant region, and

(ii) two different chimeric light chains, the first one of whichcomprises a non-human variable region having the same specificity as thefirst heavy chain variable region, and a human constant region, and thesecond comprises a non-human variable region having the same specificityas the second heavy chain variable region, and a human constant region.

The resulting antibody molecule binds to two different antigens.

The invention also provides for the production of functionally activechimeric immunoglobulin fragments secreted by prokaryotic or eukaryotichosts or fully folded and reassembled chimeric immunoglobulin chains.

Genetic sequences, especially cDNA sequences, coding for theaforementioned combinations of chimeric chains or of non-chimeric chainsare also provided herein.

The invention also provides for a genetic sequence, especially a cDNAsequence, coding for the variable region of an antibody molecule heavyand/or light chain, operably linked to a sequence coding for apolypeptide different than an immunoglobulin chain (e.g., an enzyme).These sequences can be assembled by the methods of the invention, andexpressed to yield mixed-function molecules.

The use of cDNA sequences is particularly advantageous over genomicsequences (which contain introns), in that cDNA sequences can beexpressed in bacteria or other hosts which lack RNA splicing systems.

Among preferred specific antibodies are those having specificities tocancer-related antigens.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA rearrangements and the expression of immunoglobulinmu and gamma heavy chain genes. This is a schematic representation ofthe human heavy chain gene complex, not shown to scale. Heavy chainvariable V region formation occurs through the joining of V_(H), D andJ_(H) gene segments. This generates an active mu gene. A different kindof DNA rearrangement called "class switching" relocates the joinedV_(H), D and J_(H) region from the mu constant C region to another heavychain C region (switching to gamma is diagrammed here). The schemeemphasizes that the J region is a common feature of all expressed heavychain genes. The J region is also a common feature of expressed lightchain genes.

FIG. 2 shows the known nucleotide sequences of human and mouse Jregions. Consensus sequences for the J regions are shown below theactual sequences. The oligonucleotide sequence below the mouse kappa Jregion consensus sequence is a Universal Immunoglobulin Gene (UIG)oligonucleotide which is used in the present invention.

FIG. 3 shows a scheme noting the use of the UIG oligonucleotide primerfor the synthesis of cDNA complementary to the variable region ofimmunoglobulin messenger RNA, or the use of oligo-dT as a primer forcDNA synthesis, followed by in vitro mutagenesis.

FIG. 4 shows the synthesis and analysis of human IgG1 genes, includingthree isolated clones (A.b), one of which (pGMH-6) is utilized as acloning vector (B). A 1.5 kb deletion of pBR322 sequence between Bam HIand PvuII is marked. Not to scale.

FIG. 5 shows the cloning vector pQ23, a modified pBR322, useful for cDNAcloning at the KpnI site. This vector also contains the usefulrestriction enzyme sites BglII plus SalI. Not to scale.

FIG. 6 shows in A. the synthesis and analysis of human light chain kappagenes. The Figure also shows in B. (not in scale) construction of ahuman C_(K) region cloning vector pING2001.

FIG. 7 shows primers designed for immunoglobulin V region synthesis. (A)shows the heavy chain J-C regions and primers. A DNA version of eachmouse J heavy region is shown directly above primers designed from thatsequence. Mouse J regions are 5' to 3', left to right, while primers are3' to 5', left to right. Primer names are included in brackets, andnumbers of nucleotides (N) and number of mismatches with each J_(H)region are listed to the right. Primers which introduce a BstEII siteare underlined. (B) shows the light chain J regions and primers. Thesame as for (A) except for light chains. Primers designed to introduce aBGlII site are underlined, as is the BclI site present in pING2016E. (C)shows mouse variable region consensus UIG primers. The actual primersequence is shown below the consensus sequence. The human C_(K) HindIIIvector pGML60 is shown below. (D) shows a mouse gamma 2a J/C junctionprimer.

FIG. 8 shows the synthesis of heavy chain V region module genes usingoligonucleotide primed cDNA synthesis. Not to scale.

FIG. 9 shows the construction of hybrid mouse-human immunoglobulingenes. Panel A shows construction of a heavy chain gene. Panel B showsconstruction of a light chain gene. Stippled regions show C regionmodules, while hatched or black regions show V region modules. Not toscale.

FIG. 10 shows the construction of cDNA cloning-expression shuttlevectors for mammalian cells. The vectors pING2003 and pING2003E arederived from pL1, pUC12, pSV2-neo and M8-alphaRX12. Stippled regionsindicate mouse heavy chain enhancer DNA, hatched regions indicate SV-40DNA from pL1, and cross-hatched regions indicate SV-40 DNA frompSV2-neo. In the vectors pING2003 and pING2003E, thick lines representpBR322 DNA from pSV2-neo, while thin lines represent pUC12 DNA. Arrowsindicate the locations and directions of SV-40 early region promoters,and indicates a complete SV-40 intron sequence. Not to scale. FIG. 11shows the construction of the heavy chain expression plasmid pING2006E.Arrows show SV-40 promoter locations and directions of transcription.Hatched and black areas show mouse V region modules, while stippledareas show human C region modules. Not to scale.

FIG. 12 shows the structure of the chimeric anti-hepatitis heavy chaingenes in the expression plasmids pING2006E and pING2012E. Panel A showsthe structure of mouse-human chimeric anti-hepatitis heavy chain genes.The structure of human IgG1 mRNA and cDNA is shown in A.a. The humanheavy chain constant region cDNA clone pGMH-6 and the mouse heavy chainvariable region cDNA clones pBS13-1 and pJ3-11 were used to make thehybrid gene used in pING2006E. Hatched gene blocks indicate mousevariable region sequences, while open gene blocks show human IgG1constant region sequences. Panel B shows the nucleotide sequence of theanti-hepatitis B heavy chain variable region in pING2006E and pING2012E.pING2012E was constructed by first inserting a BglII site at the SalIsite of pING1202 (See FIG. 16) to form pING1202BglII. The chimeric heavychain gene from this plasmid was inserted into the expression vectorpING2003E, resulting in pING2012E. pING2012E differs from pING 2006E inthe region immediately upstream of the initiator ATG. Underlinednucleotides denote human J region sequences from the cDNA clone pGMH-6.Asterisked amino acid 117 indicates a single change at this site frommouse to human sequence (Ala to Ser) introduced in the chimeric gene Jregion. Sequencing was by the Sanger method on plasmid (open circle) andM13 (closed circle) templates.

FIG. 13 shows in panel A the J-C Junction region nucleotide sequence inlight chain clones derived from pING2001 (pMACK-3, pING2013E, pING2007E,pING2010E-gpt and pING2014E-gpt). The J region sequence originating frompK2-3 is marked human JK4. The G nucleotide not predicted by genomicsequencing is marked with an asterisk. The oligonucleotide primer(K2-4BCLI) used to modify this sequence is shown below the human JK4sequence. Panel B diagrams the method of site-directed mutagenesis usedto make pING2016E-gpt. Not to scale.

FIG. 14 Gene copy number of the transfected sequences in twotransformants. nDNA from 2AE9, 2BH10 were digested with the enzymesindicated. The concentration of DNA is titrated down across the laneswith the amount indicated above them. The probe contains human C gamma 1sequences (pmvHc24 ApaI-BamHI). The reference is germ-line or GM2146nDNA digested with ApaI. The 3' ApaI site is 2 bp beyond the site ofpoly(A) addition.

FIG. 15 shows the nucleotide sequence of the V region of the L6 V_(H)cDNA clone pH3-6a. The sequence was determined by the dideoxyterminationmethod using M13 subclones of gene fragments (shown below). Open circlesdenote amino acid residues confirmed by peptide sequence. A sequencehomologous to D_(SP).2 in the CDR3 region is underlined.

FIG. 16 shows the nucleotide sequence of the V region of the L6 V_(K)cDNA clone pL3-12a. The oligonucleotide primer used for site-directedmutagenesis is shown below the J_(K) 5 segment. Open circles denoteamino acid residues confirmed by peptide sequence.

FIG. 17 shows the construction of chimeric L6-V_(H) plus human C gamma 1expression plasmids. Panel (a) shows the sequences of the BAL-31deletion clones M13mp19-Cl1-delta 4 (Cl-delta 4) and M13mp19-Cl1-delta1(Cl1-delta 21). The 5' end of the cDNA clone, pH3-6a, is denoted withan arrow. M13 sequences are underlined. The oligonucleotide primer usedfor this experiment is H3-6a (5'- GACTGCACCAACTGG-3'), which primes inFR1 near the mature N terminus. Panel (b) shows the strategy forsite-directed mutagenesis of 1 ug of clones Cl-delta 4 and Cl-delta 21,each annealed to 20 ng of the 31-met oligonucleotide MJH2-ApaI.Complementary strand synthesis with the Klenow fragment of DNApolymerase was at room temperature for 30 min, then 15° C. for 72 hours.Transfected phage plaques were adsorbed to nitrocellulose, fixed withNaOH, and hybridized to ³² P-labelled MJH2-ApaI oligonucleotide at 65°C., 18 hours, in 4×TBS (0.6M NaCl, 0.04M Tris-HCl (pH 7.4), 0.004M EDTA)plus 10% dextran sulfate Final wash of the filters was at 65° C.,4×SSPE, 0.1% SDS for 15 min. (Maniatis, T., et al., Molecular Cloning: ALaboratory Manual, 1982). Positive plaques were detected by overnightexposure to Kodak XAR film, and were directly picked for growth andrestriction enzyme analysis of RF DNA. Mismatches of the MJH2-ApaIoligonucleotide to the mouse C_(H) 1 are denoted, resulting in thecoding changes shown below the oligonucleotide. Panel (c) shows thestrategy of the substitution of each of the mutagenized L6-V_(H) modulesfor the resident V_(H) of the chimeric expression plasmid pING2012 togenerate pING2111 and pING2112.

FIG. 18 shows the construction of the chimeric L6 expression plasmidpING2119. The SalI to BamHI fragment from pING2100 is identical to theSalI to BamHI A fragment from pING2012E.

FIG. 19 shows the modification of the V_(K) gene and its use inconstructing light chain and heavy plus light chain expression plasmids.

(a) Deletion of the oligo d[GC] segment 5' of V_(K) of L6. Theoligonucleotide is a 22-mer and contains a SalI site. The 3 mismatchesare shown. The V_(K) gene, after mutagenesis, is joined as aSalI-HindIII fragment to the human C_(K) module. The expression plasmidthus formed is pING2119.

(b) pING2114, a heavy plus light chain expression plasmid. Theexpression plasmid pING2114 contains the L6 heavy chain chimeric genefrom pING2111 and the chimeric light chain from pING2119 (bold line).

FIG. 20 shows a summary of the sequence alterations made in theconstruction of the L6 chimeric antibody expression plasmids. Residuesunderlined in the 5' untranslated region are derived from the clonedmouse kappa and heavy- chain genes. Residues circled in the V/C boundaryresult from mutagenesis operations to engineer restriction enzyme sitesin this region. Residues denoted by small circles above them in the L6heavy-chain chimera also result from mutagenesis. They are silentchanges.

FIG. 21 shows the 2H7 V_(H) sequence. The V_(H) gene contains J_(H) 1sequences and DSP.2 sequence elements. Small circles above the aminoacid residues are those that matched to peptide sequences.

FIG. 22 shows the 2H7 V_(L) sequence. The V_(K) gene contains J_(K) 5sequences. A 22-mer oligonucleotide was used to place a SalI site 5' ofthe ATG initiator codon. Small circles above the amino acid residues arethose that matched to peptide sequences.

FIG. 23 shows the chimeric immunoglobulin gene expression plasmids ofthe 2H7 specificity. One gene plasmids are pING2101 (V_(H),neo),pING2106 (V_(K),neo) and pING2107 (V_(K),gpt). The others are two-geneplasmids. Their construction involved the ligation of the larger NdeIfragments of pING2101 and pING2107 to linearized pING2106 partiallydigested with NdeI. pHL2-11 and pHL2-26 were obtained from pING2101 andpING2106; pLL2-25 was obtained from pING2107 and pING2106.

FIG. 24 shows a summary of the nucleotide changes introduced in theV_(H) and V_(K) in the construction of the chimeric plasmids. Thecognate V_(H) and V_(K) nucleotide residues in the 5' end areunderlined. Circles residues in the J-C junctions are derived from thehuman C modules.

FIG. 25 shows the strategy used to fuse the mature L6 chimeric lightchain sequence to the yeast invertase signal sequence and shortened PGKpromoter. The open double line represents yeast invertase signalsequence DNA. The solid double line represents yeast PGK DNA;->represents the PGK promoter; -| represents the PGK terminator;RF=Replicative Form. pING1225 was derived by fusing human CK DNA to thePGK promoter. pING1149 was derived by fusing the yeast invertase signalsequence to the yeast PGK promoter. (A) shows the strategy forintroduction by in vitro mutagenesis of an AatII site in the signalsequence processing site. (B) shows the DNA sequence of thesingle-stranded mutagenesis primer and the corresponding unmutagenizedDNA sequence. (C) shows the strategy used to construct a plasmidcontaining the mature light chain sequence fused to the invertase signalsequence and shortened PGK promoter.

FIG. 26 shows the strategy used to fuse the mature L6 chimeric heavychain sequence to the yeast invertase signal sequence and shortened PGKpromoter. pING1288 contains the chimeric heavy chain gene with thevariable region from the 2H7 mouse monoclonal antibody (see example IV).All symbols are as defined in legend for FIG. 25. (A) shows the strategyfor introduction by in vitro mutagenesis of an SstI site in the signalsequence processing site. (B) shows the DNA sequence of thesingle-stranded mutagenesis primer and the corresponding unmutagenizedDNA sequence. (C) shows the strategy used to construct a plasmidcontaining the mature heavy chain sequence fused to the invertase signalsequence and shortened PGK promoter.

FIG. 27 shows the strategy used to remove non-yeast 3' untranslated DNAsequences from the L6 chimeric light chain gene and to construct aplasmid containing the light chain gene fused to the invertase signalsequence and shortened PGK promoter in which all sequences are eitherknown by DNA sequence analysis or proven to be functional. pBR322NA isderived from pBR322 by deletion of DNA from NdeI to AuaI. Symbols are asdefined in legend for FIG. 25.

FIG. 28 shows the strategy used to remove non-yeast 3' untranslated DNAsequence from the L6 chimeric heavy chain gene and to construct aplasmid containing the heavy chain gene fused to the invertase signalsequence and shortened PGK promoter in which all sequences are eitherknown by DNA sequence analysis or proven to be functional. Symbols areas defined in legend for FIG. 25.

FIG. 29 shows the strategy used to clone the L6 chimeric light chaingene fused to the invertase signal sequence and shortened PGK promoterinto yeast-E. coli shuttle vectors containing the PGK transcriptiontermination-polyadenylation signal, yeast replication sequences, andgenes for selection of transformants. Symbols are as defined in legendfor FIG. 25.

FIG. 30 shows the strategy used to clone the L6 chimeric heavy chaingene fused to the invertase signal sequence and shortened PGK promoterinto yeast-E. coli shuttle vectors containing the PGK transcriptiontermination-polyadenylation signal, yeast replication sequences, andgenes for selection of transformants. Symbols are as defined in legendfor FIG. 25.

FIG. 31 shows a schematic diagram of the structure of human IgG1.

FIG. 32(A) shows the strategy used to introduce a stop codon and BclIsite into the hinge region of human gamma 1. (B) shows the DNA sequenceof the single-stranded primer used for in vitro mutagenesis of thegamma-1 hinge region and the corresponding unmutagenized sequence.Vertical arrows represent inter-chain disulfide bonds. Symbols are asdefined in legend for FIG. 25.

FIG. 33 shows the strategy used to fuse the L6 chimeric heavy chain genecontaining a stop codon in the hinge region (Fd chain) to the yeastinvertase signal sequence and shortened PGK promoter. Symbols are asdefined in legend for FIG. 25.

FIG. 34 shows the strategy used to remove non-yeast 3' untranslatedsequences from the L6 chimeric Fd chain and to construct a plasmidcontaining the Fd chain fused to the invertase signal sequence andshortened PGK promoter in which all sequences are either known by DNAsequence analysis or proven to be functional. Symbols are as defined inlegend for FIG. 25.

FIG. 35 shows the strategy used to clone the L6 chimeric Fd chain genefused to the invertase signal sequence and shortened PGK promoter intoyeast-E. coli shuttle vectors containing the PGK transcriptiontermination-polyadenylation signal, yeast replication sequence, andgenes for selection of transformants. Symbols are as defined in legendfor FIG. 25.

FIG. 36(A) shows the nucleotide sequence surrounding the N-terminus ofthe Erwinia caratovora pelB gene (Lei, S. P., et al., J. Bacteriol.(1987, in press)). The NdeI and HaeIII sites used in cloning are shown.The arrow indicates the leader peptidase cleavage site for pectatelyase. (B) shows the cloning strategy for construction of the pelBleader cartridge. pSS1004 contains a 1.9 kb DraI fragment cloned intothe SmaI site of pUC8. Symbols are defined in the legend for FIG. 39.

FIG. 37 shows the construction of light chain expression plasmidspRR177-8, pRR180, pRR190, and pRR191. In addition to the plasmidsdescribed in the text, M13mp18 and pIT181 were used. pIT181 contains themature light chain gene fused directly following the ATG initiationcodon of the araB gene in pIT2 (see FIG. 40).

FIG. 38 shows the construction of Fd expression plasmids pRR178-5,pRR186, and pRR196.

FIG. 39 shows the restriction maps of the light chain and Fd genecassette in pFK100, pFK101, pFK102, pFK103, and pFK104. These plasmidswere constructed as described in the text using plasmids outlined inFIG. 37 and 38. The arrow indicates the direction of transcription fromthe lac promoter. E. caratovora and eukaryotic non-coding sequences areshown as open bars. The pelB leader sequence is cross-hatched and theclosed bar represents the antibody genes Fd and light chain (K).

FIG. 40(A) shows the construction of a vector for arabinose inducibleFab expression. Plasmid pIT2 (Mason and Ray, Nucl. Acids Res. 14:5693(1986)) is a 6431 bp plasmid encoding the araC gene, the araB promoter,and a portion of the araB gene from pING1 (Johnston, S., et al., Gene34:134 (1985)) in a derivative of pBR322. An NcoI site has beenengineered at the ATG initiation codon of the araB gene. (B) shows theintroduction of the laci gene into pFK102.

FIG. 41 outlines the procedures used to create plasmids pSS1004,containing the pelB gene, and pSS1038, containing the pelB gene undercontrol of the araBAD promoter.

FIG. 42 outlines the procedure used in construction of the plasmidpING177-1, containing the pelB signal sequence and the plant thaumatingene under control of the araBAD promoter.

FIG. 43 outlines the procedures followed in construction of plasmidpING177-3, containing the plant thaumatin gene with the pre-thaumatinleader peptide sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS INTRODUCTION

Generally, antibodies are composed of two light and two heavy chainmolecules. Light and heavy chains are divided into domains of structuraland functional homology. The variable regions of both light (V_(L)) andheavy (V_(H)) chains determine recognition and specificity. The constantregion domains of light (C_(L)) and heavy (C_(H)) chains conferimportant biological properties such as antibody chain association,secretion, transplacental mobility, complement binding, and the like.

A complex series of events leads to immunoglobulin gene expression in Bcells. The V region gene sequences conferring antigen specificity andbinding are located in separate germ line gene segments called V_(H), Dand J_(H) ; or V_(L) and J_(L). These gene segments are Joined by DNArearrangements to form the complete V regions expressed in heavy andlight chains respectively (FIG. 1). The rearranged, joined (V_(L) -J_(L)and V_(H) -D-J_(H)) V segments then encode the complete variable regionsor antigen binding domains of light and heavy chains, respectively.

DEFINITIONS

Certain terms and phrases are used throughout the specification andclaims. The following definitions are provided for purposes of clarityand consistency.

1. Expression vector--a plasmid DNA containing necessary regulatorysignals for the synthesis of mRNA derived from gene sequences, which canbe inserted into the vector.

2. Module vector--a plasmid DNA containing a constant or variable regiongene module.

3. Expression plasmid--an expression vector that contains an insertedgene, such as a chimeric immunoglobulin gene.

4. Gene cloning--synthesis of a gene, insertion into DNA vectors, andidentification by hybridization and the like.

5. Transfection--the transfer of DNA into mammalian cells.

6. Promoter region--a nucleotide sequence which provides a cell with theregulatory sequences needed to express an operably linked cistron oroperon.

7. Secretion signal--a polypeptide present at the N-terminus of achimeric immunoglobulin chain useful in aiding in the secretion of thechain to the outside of the host. Also called "leading peptide," or"leader."

GENETIC PROCESSES AND PRODUCTS

The invention provides a novel approach for the cloning and productionof human antibodies with desired specificity. Generally, the methodcombines five elements:

(1) Isolation of messenger RNA (mRNA) from B cell hybridoma linesproducing monoclonal antibodies against specific antigens, cloning andcDNA production therefrom;

(2) Preparation of Universal Immunoglobulin Gene (UIG) oligonucleotides,useful as primers and/or probes for cloning of the variable region genesegments in the light and heavy chain mRNA from specific human ornon-human hybridoma cell lines, and cDNA production therefrom;

(3) Preparation of constant region gene segment modules by cDNApreparation and cloning, or genomic gene preparation and cloning;

(4) Construction of complete heavy or light chain coding sequences bylinkage of the cloned specific immunoglobulin variable region genesegments of part (2) above to cloned human constant region gene segmentmodules;

(5) Expression and production of light and heavy chains in selectedhosts, including prokaryotic and eukaryotic hosts, either in separatefermentations followed by assembly of antibody molecules in vitro, orthrough production of both chains in the same cell.

The invention employs cloned hybridoma B cell lines producing monoclonalantibodies of defined specificity for the isolation of mRNA for cDNAcloning. Because many lymphoid cell lines contain highly activenucleases which degrade mRNA during isolation, the invention uses mRNApreparation methods specifically developed for the isolation of intactmRNA from cells and tissues containing active nucleases. One such methodyields total RNA preparations by cell or tissue disruption is anethanolperchlorate dry ice mixture which reduces nuclease action(Lizardi, P. M. et al., Anal. Biochem., 98: 116 (1979)). This methodgives intact translatable mRNA.

Other methods that have been used for this invention include extractionof cells with lithium chloride plus urea (Auffray, C., and Rougeon, F.,Eur. J. Biochem., 107: 303 (1980)) or guanidine thiocyanate (Chirgwin,J. M. et al., Biochemistry, 18: 5294 (1979)) to prepare total RNA.

One universal feature of all expressed immunoglobulin light and heavychain genes and messenger RNAs is the so-called J region (i.e. joiningregion, see FIG. 1). Heavy and light chain J regions have differentsequences, but a high degree of sequence homology exists (greater than80%) within the heavy J_(H) regions or the kappa light chain J regions.The invention provides consensus sequences of light and heavy chain Jregions useful in the design of oligonucleotides (designated herein asUIGs) for use as primers or probes for cloning immunoglobulin light orheavy chain mRNAs or genes (FIGS. 2 or 7). Depending on the nature ofdesign of a particular UIG, it may be capable of hybridizing to allimmunoglobulin mRNAs or genes containing a single specific J sequence,such as UIG-MJH3 which detects only mouse J_(H) 3 sequences (FIG. 7).

Another utility of a particular UIG probe may be hybridization to lightchain or heavy chain mRNAs of a specific constant region, such asUIG-MJK which detects all mouse J_(K) containing sequences (FIG. 7). UIGdesign can also include a sequence to introduce a restriction enzymesite into the cDNA copy of an immunoglobulin gene (see FIG. 7). Theinvention may, for example, utilize chemical gene synthesis to generatethe UIG probes for the cloning of V regions immunoglobulin mRNA fromhybridoma cells making monoclonal antibodies of desired antigenspecificities.

A multi-stage procedure is utilized for generating complete V+C regioncDNA clones from hybridoma cell light and heavy chain mRNAs. In thefirst stage, the invention utilizes UIG probes as "primers" for reversetranscriptase copying of the complete V region and leader codingsequences of heavy and light chain mRNAs (FIG. 3). The complementarystrand of the primer extended cDNA is then synthesized, and thisdouble-stranded cDNA is cloned in appropriate cDNA cloning vectors suchas pBR322 (Gubler and Hoffman, Gene, 25: 263 (1983)) or pQ23 (FIG. 5;Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Publications, New York, page 224 (1982)). Clones arescreened for specific hybridization with UIG oligonucleotide probes.Positive heavy and light chain clones identified by this screeningprocedure are mapped and sequenced to select those containing V regionand leader coding sequences.

An alternative method is to make cDNA clones using oligo-dT as a primer,followed by selection of light and heavy chain clones by standardhybridization methods.

A second stage utilizes cloning of C region gene segments to form heavyand light chain module vectors. In one method cDNA clones of human heavyand light chain immunoglobulin mRNA are prepared. These cDNA clones arethen converted into C region module vectors by site-directed mutagenesisto place a restriction site at a desired location near a boundary of theconstant region. An alternative method utilizes genomic C region clonesas the source for C region module vectors.

A third stage of cDNA cloning involves the generation of complete lightand heavy chain coding sequences with linked V and C regions. The clonedV region segments generated as above are excised and ligated to light orheavy chain C region module vectors. For example, one can clone thecomplete human kappa light chain C region and the complete human gamma₁C region. In addition, one can modify a human gamma 1 region andintroduce a termination codon, and thereby obtain a gene sequence whichencodes the heavy chain portion of an Fab molecule.

The coding sequences having operationally linked V and C regions arethen transferred into appropriate expression systems for expression inappropriate hosts, prokaryotic or eukaryotic. Operationally linked meansin-frame joining of coding sequences to derive a continuouslytranslatable gene sequence without alterations or interruptions of thetriplet reading frame.

One particular advantage of using cDNA genetic sequences in the presentinvention is the fact that they code continuously for immunoglobulinchains, either heavy or light. By "continuously" is meant that thesequences do not contain introns (i.e. are not genomic sequences, butrather, since derived from mRNA by reverse transcription, are sequencesof contiguous exons). This characteristic of the cDNA sequences providedby the invention allows them to be expressible in prokaryotic hosts,such as bacteria, or in lower eukaryotic hosts, such as yeast.

Another advantage of cDNA cloning methods is the ease and simplicity ofobtaining V region gene modules.

The term "non-human" as used in the invention is meant to include anyanimal other than a human, wherein an immune response can be generatedwhich then leads to usable B cells resulting in corresponding hybridomasor B cell clones obtained by vital transformation and the like. Suchanimals commonly-include rodents such as the mouse or the rat. Becauseof ease of preparation and great availability, the mouse is at presentthe preferred, non-human animal. Mouse-mouse hybridomas are thusutilized as the preferred sources for heavy and light chain variableregions.

Preferably, the invention provides entire V and/or C region cDNAsequences. This means that the sequences code for substantially operableV and/or C regions, without lacking any major structural portionsthereof.

The terms "constant" and "variable" are used functionally to denotethose regions of the immunoglobulin chain, either heavy or light chain,which code for properties and features possessed by the variable andconstant regions in natural non-chimeric antibodies. As noted, it is notnecessary for the complete coding region for variable or constantregions to be present, as long as a functionally operating region ispresent and available.

A wide range of source hybridomas are available for the preparation ofmRNA. For example, see the catalogue ATCC CELL LINES AND HYBRIDOMAS,December, 1984, American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A., pages 5-9 and the ECACC Catalogue, 2ndEdition; PHLS CAMR Porton Down, Salisbury, Wills; SP4OJG, U.K. pages30-35 and 40-46. Hybridomas secreting monoclonal antibodies reactive toa wide variety of antigens are listed therein, are available from thecollection, and usable in the invention. Of particular interest arehybridomas secreting antibodies which are reactive with vital antigens,including Dengue complex specific (ATCC HB 114), Dengue type 1 virus(ATCC HB 47), Dengue type 2 virus (ATCC HB 46), Dengue type 3 virus(ATCC HB 49), Dengue type 4 virus (ATCC HB 48), Epstein-Barr receptor(ATCC HB 135), Flavivirus group (ATCC HB 112), hepatitis B surfaceantigen (ATCC CRL 8017 and 8018), herpes simplex type I (ATCC HB 8068),herpes simplex type II (ATCC HB 8067), influenza virus (ATCC CL 189),influenza A virus, matrix protein (ATCC HB 64), influenza A virus,nucleoprotein (ATCC HB 65), influenza A Bangkok/1/79HA (ATCC HB 66),influenza AWSN NP (ATCC HB 67), SV40 large T antigen (ATCC TIB 115),SV40 large T antigen, C-terminal end (ATCC TIB 117), and SV40 nonviral Tantigen (ATCC TIB 116). Examples of other hybridomas include thosesecreting antibodies to tumor associated antigens or to human lymphocyteantigens, such as those reactive to human tumor-associated CEA, high mw(ATCC CRL 8019); human tumor-associated alpha-fetoprotein, IgG₁ K (ATCCHB 134); human B lymphocyte HLA-DR, monomorphic, IgG_(2b) (ATCC HB 104);human T lymphocyte T cell precursors, IgG₁ (ATCC CRL 8022); human Tlymphocyte T cell subset, helper, IgG_(2b) (ATCC CRL 8002); T subset,suppressor/cytotoxic, human, IgG₁ (ATCC CRL 8013); T cell subset,suppressor/cytotoxic, human, IgG_(2a) (ATCC CRL 8014); T cells,peripheral, human, IgG₁ (ATCC CRL 8000); T cells, peripheral, human,IgG_(2a) (ATCC CRL 8001); thymocytes, "common," human IgG₁ (ATCC CRL8020).

These lines and others of similar nature can be utilized to copy themRNA coding for variable region, using the UIG probes. Of particularinterest are antibodies with specificity to human tumor antigens.

Expression vehicles include plasmids or other vectors. Preferred amongthese are vehicles carrying a functionally complete human constant heavyor light chain sequence having appropriate restriction sites engineeredso that any variable heavy or light chain sequence with the appropriatecohesive ends can be easily inserted thereinto. Human constant heavy orlight chain sequence-containing vehicles are thus an importantembodiment of the invention. These vehicles can be used as intermediatesfor the expression of any desired complete heavy or light chain in anyappropriate host.

One preferred host is yeast. Yeast provides substantial advantages forthe production of immunoglobulin light and heavy chains. Yeasts carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forovert production of the desired proteins in yeast. Yeast recognizesleader sequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e. prepeptides) (Hitzman, et al., 11thInternational Conference on Yeast, Genetics and Molecular Biology,Montpelier, France, September 13-17, 1982).

Yeast gene expression systems can be routinely evaluated for the levelof heavy and light chain production, protein stability, and secretion.Any of a series of yeast gene expression systems incorporating promoterand termination elements from the actively expressed genes coding forglycolytic enzymes produced in large quantities when yeasts are grown inmediums rich in glucose can be utilized. Known glycolytic genes can alsoprovide very efficient transcription control signals. For example, thepromoter and terminator signals of the iso-1-cytochrome C (CYC-1) genecan be utilized.

The following approach can be taken for evaluating optimal expressionplasmids for the expression of cloned immunoglobulin. cDNAs in yeast.

(1) The cloned immunoglobulin DNA linking V and C regions is attached todifferent transcription promoters and terminator DNA fragments;

(2) the chimeric genes are placed on yeast plasmids used for proteinoverproduction (see, for example, Beggs, J. D., Molecular Genetics andYeast, Alfred Benzon Symposium, 16, Copenhagen (1981));

(3) Additional genetic units such as a yeast leader peptide may beincluded on immunoglobulin DNA constructs to obtain antibody secretion.

(4) A portion of the sequence, frequently the first 6 to 20 codons ofthe gene sequence may be modified to represent preferred yeast codonusage.

(5) The chimeric genes are placed on plasmids used for integration intoyeast chromosomes.

The following approaches can be taken to simultaneously express bothlight and heavy chain genes in yeast.

(1) The light and heavy chain genes are each attached to a yeastpromoter and a terminator sequence and placed on the same plasmid. Thisplasmid can be designed for either autonomous replication in yeast orintegration at specific sites in the yeast chromosome.

(2) The light and heavy chain genes are each attached to a yeastpromoter and terminator sequence on separate plasmids containingdifferent selective markers. For example, the light chain gene can beplaced on a plasmid containing the trp1 gene as a selective marker,while the heavy chain gene can be placed on a plasmid containing ura3 asa selective marker. The plasmids can be designed for either autonomousreplication in yeast or integration at specific sites in yeastchromosomes. A yeast strain defective for both selective markers iseither simultaneously or sequentially transformed with the plasmidcontaining light chain gene and with the plasmid containing heavy chaingene.

(3) The light and heavy chain genes are each attached to a yeastpromoter and terminator sequence on separate plasmids each containingdifferent selective markers as described in (2) above. A yeast matingtype "a" strain defective in the selective markers found the light andheavy chain expression plasmids (trp1 and ura3 in the above example) istransformed with the plasmid containing the light chain gene byselection for one of the two selective markers (trp1 in the aboveexample). A yeast mating type "alpha" strain defective in the sameselective markers as the "a" strain (i.e. trp1 and ura3 as examples) istransformed with a plasmid containing the heavy chain gene by selectionfor the alternate selective marker (i.e. ura3 in the above example). The"a" strain containing the light chain plasmid (phenotype: Trp⁺ Ura⁻ inthe above example) and the strain containing the heavy chain plasmid(phenotype: Trp⁻ Ura⁺ in the above example) are mated and diploids areselected which are prototrophic for both of the above selective makers(Trp⁺ Ura⁺ in the above example).

Among bacterial hosts which may be utilized as transformation hosts, E.coli K12 strain 294 (ATCC 31446) is particularly useful. Other microbialstrains which may be used include E. coli X1776 (ATCC 31537). Theaforementioned strains, as well as E. coli W3110 (ATCC 27325) and otherenterobacteria such as Salmonella typhimurium or Serratia marcescens,and various Pseudomonas species may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with a host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as specific genes which are capable of providingphenotypic selection in transformed cells. For example, E. coli isreadily transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene, 2: 95 (1977)). pBR322 contains genes forampicillin and tetracycline resistance, and thus provides easy means foridentifying transformed cells. The pBR322 plasmid or other microbialplasmids must also contain, or be modified to contain, promoters whichcan be used by the microbial organism for expression of its ownproteins. Those promoters most commonly used in recombinant DNAconstruction include the beta-lactamase (penicillinase) and lactose(beta-galactosidase) promoter systems (Chang et al., Nature, 275: 615(1978); Itakura et al., Science, 198: 1056 (1977)); and tryptophanpromoter systems (Goeddel et al., Nucleic Acids Research, 8: 4057(1980); EPO Publication No. 0036776). While these are the most commonlyused, other microbial promoters have been discovered and utilized.

For example, a genetic construct for any heavy or light chimericimmunoglobulin chain can be placed under the control of the leftwardpromoter of bacteriophage lambda (P_(L)). This promoter is one of thestrongest known promoters which can be controlled. Control is exerted bythe lambda repressor, and adjacent restriction sites are known.

The expression of the immunoglobulin chain sequence can also be placedunder control of other regulatory sequences which may be "homologous" tothe organism in its untransformed state. For example, lactose dependentE. coli chromosomal DNA comprises a lactose or lac operon which mediateslactose digestion by elaborating the enzyme beta-galactosidase. The laccontrol elements may be obtained from bacteriophage lambda pLAC5, whichis infective for E. coli. The lac promoter-operator system can beinduced by IPTG.

Other promoter/operator systems or portions thereof can be employed aswell. For example, arabinose, colicine El, galactose, alkalinephosphatase, tryptophan, xylose, tac, and the like can be used. Otherbacterial gene expression control elements can be utilized to achievethe expression of immunoglobulin proteins. For example, a gene with abacterial secretion signal peptide coding region can be expressed inbacteria, resulting in secretion of the immunoglobulin peptide which wasoriginally linked to the signal peptide.

Other preferred hosts are mammalian cells, grown in vitro in tissueculture, or in vivo in animals. Mammalian cells providepost-translational modifications to immunoglobulin protein moleculesincluding leader peptide removal, correct folding and assembly of heavyand light chains, glycosylation at correct sites, and secretion offunctional antibody protein from the cell as H₂ L₂ molecules.

Mammalian cells which may be useful as hosts for the production ofantibody proteins include cells of fibroblast origin, such as Vero (ATCCCRL 81) or CHOK1 (ATCC CRL 61), or cells of lymphoid origin, such as thehybridoma Sp2/0-Ag14 (ATCC CRL 1581) or the myleoma P3X63Ag8 (ATCC TIB9), and its derivatives.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors utilizes DNA elements which provide an autonomouslyreplicating extrachromosomal plasmid, derived from animal viruses, suchas bovine papillomavirus (Sarver, N. et al., Proc. Natl. Acad. Sci.,USA, 79: 7147 (1982)), polyoma virus (Deans, R. J. et al., Proc. Natl.Acad. Sci., USA, 81: 1292 (1984)), or SV40 virus (Lusky, M. and Botchan,M., Nature, 293: 79 (1981)). A second class of vectors relies upon theintegration of the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing drug resistance genessuch as E. coli gpt (Mulligan, R. C. and Berg, P., Proc. Natl. Acad.Sci., USA, 78: 2072 (1981)) or Tn5 neo (Southern, P. J. and Berg, P., J.Mol. Appl. Genet., 1: 327 (1982)). The selectable marker gene can beeither directly linked to the DNA gene sequences to be expressed, orintroduced into the same cell by co-transfection (Wigler, M. et al.,Cell, 16: 77 (1979)).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein or its precursor,additional gene expression elements regulating transcription of the geneand processing of the RNA are required for optimal synthesis ofimmunoglobulin mRNA. These elements may include splice signals, as wellas transcription promoters including inducible promoters, enhancers, andtermination signals. cDNA expression vectors incorporating such elementsinclude those described by Okayama, H. and Berg, P., Mol. Cell Biol., 3:280 (1983); Cepko, C. L. et al., Cell, 37: 1053 (1984); and Kaufman, R.J., Proc. Natl. Acad. Sci., USA, 82:689 (1985).

An approach to evaluate optimal vectors for the expression ofimmunoglobulin cDNA in mammalian cells involves first placing theimmunoglobulin DNA sequences into vectors capable of stably integratinginto the cell genome, or replicating autonomously as an extrachromosomalplasmid. The vectors can be used to evaluate different gene expressionelements for optimal immunoglobulin synthesis.

An additional advantage of mammalian cells as hosts is their ability toexpress chimeric immunoglobulin genes which are derived from genomicsequences. Thus, mammalian cells may express chimeric immunoglobulingenes which are comprised of a variable region cDNA module plus aconstant region which is composed in whole or in part of genomicsequences. Several human constant region genomic clones have beendescribed (Ellison, J. W. et al., Nucl. Acids Res., 10: 4071 (1982), orMax, E. et al., Cell, 39: 691 (1982)). The use of such genomic sequencesmay be convenient for the simultaneous introduction of immunoglobulinenhancers, splice signals, and transcription termination signals alongwith the constant region gene segment.

Different approaches can be followed to obtain complete H₂ L₂antibodies.

First, one can separately express the light and heavy chains followed byin vitro assembly of purified light and heavy chains into complete H₂ L₂IgG antibodies. The assembly pathways used for generation of complete H₂L₂ IgG molecules in cells have been extensively studied (see, forexample, Scharff, M., Harvey Lectures, 69: 125 (1974)). In vitroreaction parameters for the formation of IgG antibodies from reducedisolated light and heavy chains have been defined by Beychok, S., Cellsof Immunoglobulin Synthesis, Academic Press, New York, page 69, 1979.

Second, it is possible to co-express light and heavy chains in the samecells to achieve intracellular association and linkage of heavy andlight chains into complete H₂ L₂ IgG antibodies. The co-expression canoccur by using either the same or different plasmids in the same host.

In a preferred embodiment, the co-expression can occur with aid ofsecretion signals useful in yeast or bacteria. Under such conditions,fully folded and assembled H₂ L₂ immunoglobulins can be obtained.

The present invention provides plasmid secretion vectors which, whenused to transform bacterial host cells, enable the externalization ofexpressed foreign proteins. The plasmids comprise a DNA sequence codingfor a pectate lyase signal sequence of a gram-negative bacterium. Theseplasmids are in turn used to prepare derivative plasmids containing afragment consisting essentially of the signal sequence and a non-hostprotein gene sequence, and plasmids in which this fragment is positionedadjacent to a host-compatible promoter sequence. The latter plasmids,containing the promoter sequence, when used to transform a bacterialhost cell, are capable of directing the translation and externalizationof the foreign proteins by the host cell. The present invention thusprovides a means by which foreign proteins can be produced in highvolume by host cells without the necessity for cell lysis, and theattendant extensive purification procedures required to removecytoplasmic contaminants. Isolation of expressed non-host proteins fromthe host would therefore be significantly facilitated if they can beexternalized in this manner. The pectate lyase (pel) signal sequencefunctions well in the E. coli cell system, and the present secretionvectors have been shown to be capable of causing the secretion ofvirtually any protein used in the system. Secretion of the protein intothe periplasmic space or extracellular environment is achieved by theinsertion of a known gene sequence coding for a particular protein intothe plasmid vector at some point after the pel signal sequence, and asuitable promoter sequence and then transforming a bacterial host cellwith the secretion vector.

As disclosed herein, recombinant plasmids have been prepared which, whenused to transform bacterial host cells, permit the secretion of foreignprotein outside the cytoplasmic membrane of the host cell. The inventionis based on the observation that when pectate lyase genes are cloned andexpressed in E. coli, large quantities of pectate lyase are secretedeither into the periplasmic space or culture fluid in which thebacterium is grown. This leads to the conclusion that the pectate lyasesignal sequence is recognized and translated in a non-Erwinia bacterialsystem. When plasmids were prepared containing the signal sequence ofthe pel gene in combination with a foreign protein gene it wasdiscovered that, in the presence of a host-compatible promoter, thesignal sequence for the pel gene is capable of directing thedistribution of the protein from the cytoplasm into the periplasmicspace, or beyond the cell wall, into the extracellular environment. Thesecretion vectors of the present invention are particularly useful inthat their utility is not limited to a single strain, but rather havebeen shown to function efficiently in a wide variety of readilyavailable E. coli strains, all of which routinely secrete at least someof the recombinant protein directly into the culture medium. Theplasmids have also been shown to be operable with a broad range offoreign gene sequences, both procaryotic and eucaryotic. Further, theproteins so produced have been confirmed to be secreted in their naturaland proper configuration.

The manner of preparation of the plasmids, and their use in bacterialtransformation is set out in greater detail below.

IDENTIFICATION AND ISOLATION OF THE PECTATE LYASE GENE

Pectate lyase is one of the enzymes which catalyzes the hydrolysis ofpolygalacturonic acid, and is found in a number of phytopathogenicbacteria and fungi. Three pectate lyases, referred to as PLa, PLb andPLc, have been identified from the bacterium Erwinia carotovora, andsimilar enzymes are also known in the bacteria Erwinia chrysanthemi(Keen et al., J. Bacteriol. 168: 595-606, 1986) and Pseudomonasfluorescens, as well as the fungus Rhizoctonia solani. The genes fromboth E. carotovora (Lei et al., Gene 35: 63-70, 1985) and E.chrysanthemi (Keen et al., supra) have been isolated. In the presentcase, the pelB gene from E. carotovora was used as a source of thepectate lyase signal sequence. Plasmid pSS1004 (Lei et al., J.Bacteriol. 169: 4379-4383, 1987) contains the E. carotovora pelB gene.The restriction sites around the signal sequence were identified basedon the DNA sequence of the gene. The N-terminal amino acid sequence wasalso determined to confirm the location of the leader peptide. Treatmentof the pSS1004 plasmid with HaeIII and EcoRI restriction enzymesproduced a fragment containing the leader sequence. The gene sequenceand the corresponding amino acid sequence of the pelB signal peptide isshown in FIG. 36A. Alternatively, the peptide sequence may be chemicallysynthesized by known methods of peptide synthesis.

CONSTRUCTION OF SECRETION VECTORS

Once the fragment containing the appropriate signal sequence has beenidentified and isolated, it is then inserted into a cloning vehicle,preferably a plasmid. The present vectors are prepared in accordancewith the general principles of vector construction known in the art. Asused in the present specification and claims, "secretion" refers totransport of the protein into the periplasmic space, and "excretion"refers to the transport of the protein into the culture medium.

In order to eventually achieve transcription and translation of theinserted gene, the gene must be placed under the control of a promotercompatible with the chosen host cell. A promoter is a region of DNA atwhich RNA polymerase attaches and initiates transcription. The promoterselected may be any one which has been isolated from or is capable offunctioning in the host cell organism. For example, E. coli has numerouspromoters such as the lac or recA promoter associated with it, itsbacteriophages or its plasmids. Also, phage promoters, such as the λphage P_(L) and P_(R) promoters may be used to direct high levelproduction of the products coded for having the segments of DNA adjacentto it. The products may be natural, synthetic or recombinant.

An initiation signal is also necessary in order to attain efficienttranslation of the gene. For example, in E. coli mRNA, a ribosomebinding site includes the translational start codon (AUG or GUG) andanother sequence complementary to the bases of the 3'-end of 16Sribosomal RNA. Several of these latter sequences (Shine-Dalgarno or S-D)have been identified in E. coli and other suitable host cell types. AnySD-ATG sequence which is compatible with the host cell system can beemployed. These include, but are not limited to, the cro gene or N geneof coliphage lambda, or the E. coli tryptophan E, D, C, B or A genes.

A number of methods exist for the insertion of DNA fragments intocloning vectors in vitro. DNA ligase is an enzyme which sealssingle-stranded nicks between adjacent nucleotides in a duplex DNAchain; this enzyme may therefore be used to covalently join the annealedcohesive ends produced by certain restriction enzymes. Alternately, DNAligase can be used to catalyze the formation of phosphodiester bondsbetween blunt-ended fragments. Finally, the enzyme terminaldeoxynucleotidyl transferase may be employed to form homopolymeric3'-single-stranded tails at the ends of fragments; by addition of oligo(dA) sequences to the 3'-end of one population, and oligo (dT) blocks to3'-ends of a second population, the two types of molecules can anneal toform dimeric circles. Any of these methods may be used to ligate thegene segment, promoter and other control elements into specific sites inthe vector. Thus, the coding sequence for a particular protein Asligated into the chosen vector in a specific relationship to the vectorpromoter and control elements and to the pel signal sequence, so thatthe protein gene sequence As An the correct reading frame with respectto the vector ATG sequence. The vector employed will typically have amarker function, such as ampicillin resistance or tetracyclineresistance, so that transformed cells can be identified. The vector maybe any of the known expression vectors or their derivatives; among themost frequently used are plasmid vectors such as pBR322, pAC105, pVA5,pACYC177, PKH47, pACYC184, pUB110, pMB9, pBR325, ColE1, pSC101, pBR313,pML21, RSF2124, pCR1 or RP4; bacteriophage vectors such as lambda gt11,lambda gt-WES-lambdaB, Charon 28, Charon 4A, lambda gt-1-lambda BC,lambda-gt-1-lambda B, M13mp7, M13mp8, M13mp9; SV40 and adenovirusvectors, and yeast vectors.

The present invention preferably employs a plasmid as the cloningvector. For example, the approach taken in the present examples, in thecloning of a number of different proteins in E. coli, was to firstseparately clone the PLb signal sequence. This was achieved by isolationof the HaeIII+EcoRI digest fragment from plasmid pSS1004 which containsthe entire pectate lyase B sequence. This fragment is then ligated intoa pBR322 plasmid, which has been digested with SspI, ligated with anSstI linker, and then digested with EcoRI. Thus formed is the plasmidpING173 containing the pelB signal sequence. The pING173 plasmid is thenused as a cloning vehicle for the pelB signal sequence, which may besubsequently ligated into a plasmid containing the sequence of theprotein of interest. The plasmid so prepared contains a hybrid geneconsisting of the pelB signal sequence adjacent to the relevant proteingene sequence; an appropriate promoter sequence may then be inserted atthe 5'- terminus of the hybrid gene thus creating the final expressionvector. Alternately, a plasmid containing the promoter sequence and thepelB signal sequence can first be prepared, and then the protein genesequence may be inserted downstream of the promoter-signal sequences.Promoters which have proven particularly useful in the present inventionare the Salmonella typhimurium araBAD and E. coli lac promoters incombination with an E. coli host system. However, any other suitablepromoter which is compatible with the host system of choice may also beemployed. Prepared by the foregoing method are plasmid expressionvectors containing the genes for thaumatin, and L6 chimeric Fab, but itwill be readily apparent to one skilled in the art that genes for anytype of protein may be used in the present vectors and methods.

ISOLATION OF THE GENE PRODUCT

The present invention permits the gene product to be isolated fromeither the periplasmic space or the surrounding growth medium. Thelocation of the expressed protein appears to be dependent on theparticular strain utilized as the host. One of the most unexpectedaspects of the present invention is that all strains tested were capableof excreting at least some of the recombinant product into the culturemedium. The principal distinction between strains is the ratio of theamount of excreted product (i.e., that which is transported into themedium) to the amount secreted into the periplasmic space. Among thoseE. coli strains which show high levels of excretion are MC1061, JM103and 706. While proteins which are excreted into the culture fluid arereadily isolatable therefrom by known protein recovery techniques, therecovery of protein localized in the periplasmic space requirespenetration of the cell wall in order to achieve release of the proteinswithout disruption of the cytoplasmic membrane. One technique of removalof periplasmic proteins is that originally described by Zinder and Arndt(PNAS USA 42: 586-590, 1956), which involves removal of the cell wall.Briefly, the cells are treated with egg albumin, which containslysozyme, producing cellular spheres, on spheroplasts, which have largeportions of the cell wall removed. Periplasmic proteins may also beisolated by a mechanism which does not require removal of the cell wall,but instead causes release of the proteins. Cells are placed in ahypertonic sucrose medium containing ethylene diamine tetraacetic acid(EDTA); this medium causes the cells to lose water and shrink, so thatthe cytoplasmic membrane draws away from the cell wall. The cells arethen placed in a magnesium chloride solution which induces an osmoticshock: the osmotic pressure outside the cell decreases, causing water torush into the cell, which swells the cell and causes the expulsion ofperiplasmic proteins beyond the outer membrane. Variations in theforegoing procedures will be readily apparent to one skilled in the art.

Also, preparation of chimeric Fab fragments can be carried out by themethods of the invention.

The methods described herein can also be used to switch the class of anyantibody of a given specificity and class to an antibody of the samespecificity but of a different class, whether human or non-human. Forexample, human IgM antibodies can be transmuted to human IgG antibodiesby preparing constructs containing human constant IgG cDNA or genomicsequences, linked to variable human cDNA sequences obtained from a cellproducing the original IgM antibody. These constructs are thenintroduced into appropriate hosts and expressed.

POLYPEPTIDE PRODUCTS

The invention provides "chimeric" immunoglobulin chains, either heavy orlight. A chimeric chain contains a constant region substantially similarto that present in the heavy chain of a natural human immunoglobulin,and a variable region having any desired antigenic specificity. Thevariable region is either from human or non-human origin.

The invention also provides immunoglobulin molecules having heavy andlight chains associated so that the overall molecule exhibits desiredbinding and recognition properties. Various types of immunoglobulinmolecules are provided: monovalent, divalent, dispecific (i.e., withdifferent variable regions), molecules with chimeric heavy chains andnon-chimeric light chains, or molecules with variable binding domainsattached to peptide moieties carrying desired functions.

Antibodies having chimeric heavy chains of the same or differentvariable region binding specificity and non-chimeric (i.e., all human orall non-human) light chains, can be prepared by appropriate associationof the needed polypeptide chains. These chains are individually preparedby the modular assembly methods of the invention.

Chimeric Fab fragments are also part of this invention.

USES

The antibodies of the invention having human constant region can beutilized for passive immunization, especially in humans, withoutnegative immune reactions such as serum sickness or anaphylactic shock.The antibodies can, of course, also be utilized in prior artimmunodiagnostic assays and kits, in labelled form for in vitro imaging,wherein the label can be a radioactive emitter, or an NMR contrastingagent such as a carbon-13 nucleus, or an X-ray contrasting agent, suchas a heavy metal nucleus. The antibodies can also be used in vitrolocalization of antigens by appropriate labelling.

The antibodies can be used for therapeutic purposes by themselves incomplement mediated lysis or can be coupled to toxins or othertherapeutic moieties.

Class switching of antibodies is useful when it is desired to change theassociation, aggregation or other properties of antibodies obtained fromcell fusion or hybridoma technology. For example, most human-humanmonoclonals are of the IgM class, which are known for their ease ofreduction and aggregation. Changing such antibodies to other antibodytypes, such as IgG, IgA, or IgE, is thus of great benefit.

Mixed antibody-enzyme molecules can be used for immunodiagnosticmethods, such as ELISA. Mixed anti-body-peptide effector conjugates canbe used for targeted delivery of the effector moiety with a high degreeof efficacy and specificity.

Having now generally described the invention, the same will be furtherunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXPERIMENTAL

Materials and Methods Tissue Culture Cell Lines

The human cell lines GM2146 and GM1500 were obtained from the HumanMutant Cell Repository (Camden, N.J.) and cultured in RPMI1640 plus 10%fetal bovine serum (M. A. Bioproducts). The cell lines Sp2/0 and CRL8017 were obtained from the American Type Culture Collection and grownin Dulbecco's Modified Eagle Medium (DMEM) plus 4.5 g/l glucose (M. A.Bioproducts) plus 10% fetal bovine serum (Hyclone, Sterile Systems,Logan, Utah). Media were supplemented with penicillin/streptomycin(Irvine Scientific, Irvine, Calif.).

Recombinant Plasmid and Bacteriophage DNAs

The plasmids pBR322, pL1 and pUC12 were purchased from Pharmacia P-LBiochemicals (Milwaukee, Wis.). The plasmids pSV2-neo and pSV2-gpt wereobtained from BRL (Gaithersburg, Md.), and are available from theAmerican Type Culture Collection (Rockville, Md.). pHu-gamma-1 is asubclone of the 8.3 Kb HindIII to BamHI fragment of the human IgG1chromosomal gene. A separate isolation of the human IgG1 chromosomalgene is described by Ellison, J. W. et al., Nucl. Acids Res., 10: 4071(1982). M8alphaRX12 contains the 0.7 Kb XbaI to EcoRI fragmentcontaining the mouse heavy chain enhancer from the J-C intron region ofthe M603 chromosomal gene (Davis, M. et al., Nature, 283: 733(1979))inserted into M13mp10. G-tailed pUC9 was purchased from Pharmacia P-L.DNA manipulations involving purification of plasmid DNA by buoyantdensity centrifugation, restriction endonuclease digestion, purificationof DNA fragments by agarose gel electrophoresis, ligation andtransformation of E. coli were as described by Maniatis, T. et al.,Molecular Cloning: A Laboratory Manual, (1982). Restrictionendonucleases and other DNA/RNA modifying enzymes were purchased fromBoehringer-Mannheim (Indianapolis, Ind.), BRL, New England Biolabs(Beverly, Mass.) and Pharmacia P-L.

Oligonucleotide Preparation

Oligonucleotides were either synthesized by the triester method of Itoet al. (Nucl. Acids Res., 10: 1755 (1982)), or were purchased fromELESEN, Los Angeles, Calif. Tritylated, deblocked oligonucleotides werepurified on Sephadex-G50, followed by reverse-phase HPLC with a 0-25%gradient of acetonitrile in 10 mM triethylamine-acetic acid, pH 7.2, ona C18 uBondapak column (Waters Associates). Detritylation was in 80%acetic acid for 30 min., followed by evaporation thrice.Oligonucleotides were labeled with [gamma-³² P]ATP plus T4polynucleotide kinase.

RNA Preparation and Analysis

Total cellular RNA was prepared from tissue culture cells by the methodof Auffray, C. and Rougeon, F. (Eur. J. Biochem., 107: 303 (1980)) orChirgwin, J. M. et al. (Biochemistry, 18: 5294 (1979)). Preparation ofpoly(A)⁺ RNA, methyl-mercury agarose gel electrophoresis, and "Northern"transfer to nitrocellulose were as described by Maniatis, T. et al.,supra. Total cellular RNA or poly(A)⁺ RNA was directly bound tonitrocellulose by first treating the RNA with formaldehyde (White, B. A.and Bancroft, F. C., J. Biol. Chem., 257: 8569 (1982)). Hybridization tofilterbound RNA was with nick-translated DNA fragments using conditionsdescribed by Margulies, D. H. et al. (Nature, 295: 168 (1982))or with ³²P-labelled oligonucleotide using 4×SSC, 10×Denhardt's, 100 ug/ml salmonsperm DNA at 37° C. overnight, followed by washing in 4×SSC at 37° C.

cDNA Preparation and Cloning

Oligo-dT primed cDNA libraries were prepared from poly(A)⁺ RNA fromGM1500 and GM2146 cells by the methods of Land, H. et al. (Nucl. AcidsRes., 9: 2251 (1981)) and Gubler, V. and Hoffman, B. J., Gene, 25: 263(1983), respectively. The cDNA libraries were screened by in situhybridization (Maniatis, T., supra) With ³² P-labelled oligonucleotidesusing the conditions shown above, or with nick-translated DNA fragmentsusing the conditions of de Lange et al. (Cell, 34: 891 (1983)).

Oligonucleotide Primer Extension and Cloning

Poly(A)⁺ RNA (20 ug) was mixed with 1.2 ug primer in 40 ul of 64 mM KCl.After denaturation at 90° C. for 5 min. and then chilling in ice, 3units Human Placental Ribonuclease Inhibitor (BRL) was added in 3 ul of1M Tris-HCl, pH 8.3. The oligonucleotide was annealed to the RNA at 42°C. for 15 minutes, then 12 ul of 0.05M DTT, 0.05M MgCl₂, and 1 mM eachof dATP, dTTP, dCTP, and dGTP was added. 2 ul of alpha-³² P-dATP (400Ci/mmol, New England Nuclear) was added, followed by 3 ul of AMV reversetranscriptase (19 units/ul, Life Sciences).

After incubation at 42° C. for 105 min., 2 ul 0.5M EDTA and 50 ul 10 mMTris, 1 mM EDTA, pH 7.6 were added. Unincorporated nucleotides wereremoved by Sephadex G-50 spun column chromatography, and the RNA-DNAhybrid was extracted with phenol, then with chloroform, and precipitatedwith ethanol Second strand synthesis, homopolymer tailing with dGTP ordCTP, and insertion into homopolymer tailed vectors was as described byGubler and Hoffman, supra.

Site-Directed Mutagenesis

Single stranded M13 subclone DNA (1 ug) was combined with 20 ngoligonucleotide primer in 12.5 ul of Hin buffer (7 mM Tris-HCl, pH 7.6,7 mM MgCl₂, 50 mM NaCl). After heating to 95° C. in a sealed tube, theprimer was annealed to the template by slowly cooling from 70° C. to 37°C. for 90 minutes. 2 ul dNTPs (1 mM each), 1 ul ³² P-dATP (10 uCi), 1 ulDTT (0.1M ) and 0.4 ul Klenow DNA PolI (2u, Boehringer Mannheim) wereadded and chains extended at 37° C. for 30 minutes. To this was added 1ul (10 ng) M13 reverse primer (New England Biolabs), and theheating/annealing and chain extension steps were repeated. The reactionwas stopped with 2 ul of 0.5M EDTA, pH 8, plus 80 ul of 10 mM Tris-HCl,pH 7.6, 1 mM EDTA. The products were phenol extracted and purified bySephadex G-50 spun column chromatography and ethanol precipitated priorto restriction enzyme digestion and ligation to the appropriate vector.

Transfection of Myeloma Tissue Culture Cells

A variation of the method of Ochi, A. et al. (Nature, 302: 340 (1983))was used for protoplast fusion. 50 ml of bacteria at A₆₀₀ of 0.7 wereconverted to protoplasts by the method of Sandri-Goldin, R. M. et al.(Mol. Cell. Biol., 1: 743 (1981)), then diluted with 20 ml DMEM plus 10%FBS (final volume is 25 ml). Sp2/0 cells were harvested, pelleted at2,200×g, washed, repelleted and resuspended in DMEM at 2-5×10⁶ /ml.Bacterial protoplasts (10 ml) were mixed with 10×10⁶ Sp2/0 cells andpelleted by centrifugation at 4,000×g at 22° C. for 20 min. Afterpipetting off the supernatant, the pellet was suspended in the remainingdrop of medium by flicking the tube. 2 ml of 10% DMSO, 37% (w/v) PEG6000(Kodak) in DMEM was added dropwise with mixing over 45 sec. After 15sec., 2 ml of 42% PEG6000 in DMEM was added over 45 sec. Complete DMEM(45 ml) was slowly added with mixing. Cells were pelleted at 2500×g,then washed and pelleted thrice.

The electroporation method of Potter, H. et al. (Proc. Natl. Acad. Sci.,USA, 81: 7161 (1984)) was used. After transfection, cells were allowedto recover in complete DMEM for 48-72 hours, then were seeded at 10,000to 50,000 cells per well in 96-well culture plates in the presence ofselective medium. G418 (GIBCO) selection was at 0.8 mg/ml, mycophenolicacid (Calbiochem) was at 6 ug/ml plus 0.25 mg/ml xanthine, and HAT(Sigma) was at the standard concentration.

Assays for Immunoglobulin Synthesis and Secretion

Secreted immunoglobulin was measured directly from tissue culture cellsupernatants. Cytoplasmic protein extract was prepared by vortexing1×10⁶ cells in 160 ul of 1% NP40, 0.15M NaCl, 10 mM Tris, 1 mM EDTA, pH7.6 at 0° C., 15 minutes, followed by centrifugation at 10,000×g toremove insoluble debris.

Double antibody sandwich ELISA (Voller, A. et al., in Manual of ClinicalImmunology, 2nd Ed., Eds. Rose, N. and Friedman, H., pp. 359-371, 1980)using affinity purified antisera was used to detect specificimmunoglobulins. For detection of human IgG, the plate-bound antiserumis goat anti-human IgG (KPL, Gaithersburg, Md.) at 1/1000 dilution,while the peroxidase-bound antiserum is goat anti-human IgG (KPL orTago, Burlingame) at 1/4000 dilution. For detection of humanimmunoglobulin kappa, the plate-bound antiserum is goat anti-human kappa(Tago) at 1/500 dilution, while the peroxidase-bound antiserum is goatanti-human kappa (Cappel) at 1/1000 dilution.

Antibodies binding hepatitis B surface antigen were detected using acommercial (Abbott, AUSAB) assay.

EXAMPLES

The following examples show the preparation of chimeric antibodies eachhaving a human constant region and a non-human variable region. Theseexamples outline the step-by-step process of preparing the chimericantibodies.

EXAMPLE I Human Antibody Constant Region Gene Modules and cDNAExpression Vectors

(1) Preparation of cDNA Clones, and Vehicles Containing Same, for HeavyChain Human Constant Region

The cell line GM2146 was used as the source in mRNA preparation and cDNAcloning. This cell line secretes IgG1 (Simmons, J. G. et al., Scand. J.Immunol., 14: 1-13, 1981). Tests of this cell line indicated that itsecretes IgA as well as IgG.

The cell line was cloned, and results indicated that five of sixsubclones secreted IgG only, while one of six subclones secreted IgAonly. Poly(A)⁺ RNA was prepared from the cell line and a cDNA librarywas prepared from the poly(A)⁺ RNA by the method of Gubler, U. andHoffman, B. J., Gene, 25: 263-269 (1983). An initial plating of the cDNAtransformed into E. coli strains HB101 and RR1 yielded a total of 1500colonies, which were screened by hybridization to a HindIII to BamHIfragment of a genomic clone of human IgG1 (pHu-gamma-1). Four positiveclones were found. A fragment containing the C_(H) 3 coding region ofone of these clones, pGMH-3 (FIG. 4), was used to rescreen the originallibrary plus a new transformation of approximately 5000 colonies. Two ofthe largest clones, pGMH-6 and pGMH-15, were analyzed by restrictionenzyme digestion (FIG. 4). Both clones contained the entire constantregion of human IgG1, although it was discovered that pGMH-6 had deletedapproximately 1500 base pairs of pBR322 DNA, apparently withoutaffecting the IgG1 cDNA sequences.

Clone pGMH-6 provided the IgG1 constant region module in theconstruction of cloning vectors for heavy chain variable region cloning.

(2) Preparation of cDNA Clones, and Vehicles Containing Same, for LightChain Human Constant Region

A human cell line (GM1500) producing IgG₂ K was selected for the initialcloning phase. Poly(A)⁺ RNA prepared from GM1500 is active in in vitrotranslation using rabbit reticulocyte extracts. A cDNA library wasprepared from this RNA by the method of Land et al., Nucl. Acids Res.,9: 2251-2266 (1981), utilizing KpnI digested and dG-tailed pQ23 as thecloning vector (FIG. 5). This vector contains BglII, KpnI and SstI sitesinserted between the BamHI and SalI sites of pBR322.

In order to identify the cDNA clones generated from GM1500 RNA whichcorrespond to light chain mRNA, a DNA probe, UIG-HuK, was synthesizedand purified. The UIG-HuK oligonucleotide has the sequence5'-AGCCACAGTTCGTTT-3', and is designed to hybridize to all functionalhuman kappa mRNA species at the J-C junction. This probe was used toprime cDNA synthesis on GM1500 RNA in the presence of dideoxynucleotidesand reverse transcriptase. From 1.2 ug of total GM1500 poly(A)⁺ RNA wasused in this experiment, the entire J sequence and some of the V regionwas read, demonstrating that (1) GM1500 RNA is intact, (2) the kappaprobe is of the correct sequence, and (3) GM1500 light chain mRNAcontains J_(K) 4 sequences.

cDNA clones positive for hybridization to the light chain probe wereselected. Since the probe hybridizes to the J-C junction, the mostimportant point was to determine if the clones had complete constantregion sequence in addition to the J region.

Insert sizes for the two largest kappa cDNA clones were 0.6 and 0.9 kb;restriction enzyme mapping indicated that the entire constant regioncoding sequence was present in both clones (FIG. 6). The human kappacDNA clone pK2-3 was used to make the light chain constant region vectorpING2001 by inserting the Sau3A fragment comprising the human kappaconstant and J regions into the BclI site of pBR325 (FIG. 6B).

A variant of the human kappa cDNA clone was made by placing a HindIIIsite in the J region. This was carried out by in vitro mutagenesis usinga J_(K) HINDIII oligonucleotide primer (FIG. 7c). The resultant plasmidis pGML60.

A vector, pING2003, was constructed for the transfer and expression ofcDNA sequences in mammalian cells (FIG. 10). This vector was constructedfrom pUC12 and two plasmids containing SV40 sequences. pL1 provides anSV40 early region promoter and an SV40 late region splice sequence.pSV2-neo sequences provide a selectable marker for mammalian celltransformation and SV40 polyadenylation signal sequences. pUC12 providesa multiple cloning site for cDNA insertion.

The pING2003 vector has several useful restriction sites formodifications. These include a HindIII site useful for the insertion ofenhancer sequences, and a HindIII to XhoI fragment useful for theinsertion of alternate promoter sequences. This vector is useful in theexpression of cDNA genes in mammalian cells.

Addition of Enhancer Element to pING2003

Immunoglobulin enhancer elements have been shown to enhancetranscription of genes in their vicinity in stably transformed mousemyeloma cells by several hundred fold (Gillies, S. D. et al., Cell, 33:717, 1983; and Banerji, J. et al. Cell, 33: 729, 1983). To facilitateexpression of the mouse- human immunoglobulin genes in mouse myelomacells, the mouse immunoglobulin heavy chain enhancer element was addedto the cDNA expression vector pING2003 (FIG. 10). The mouse heavy chainenhancer region DNA was isolated from an M13 subclone of mouse heavychain genomic DNA (M8-alpha-RX12, Deans, R. J., unpublished). DNAisolated from a SalI plus EcoRI digestion of this subclone was modifiedwith HindIII linkers and inserted into the HindIII site of pING2003,resulting in the new cDNA expression vector pING2003E. This vector isuseful in the efficient expression of cDNA genes in mammalian cells,particularly mouse myeloma or hybridoma cell lines.

EXAMPLE II Human-Mouse Chimeric Anti-HBsAg Antibody Chain

(1) Preparation of cDNA Clones and Vehicles Containing Same, for HeavyChain Mouse Anti-HBsAg Variable Region.

The cell line CRL8017 was obtained from the ATCC and subcloned.Subclones were grown and tested for mouse IgG anti-hepatitis B bindingactivity using a commercially available anti-HBsAg detection kit. Threepositive subclones were found. Poly(A)⁺ RNA was prepared from one ofthese subclones, and was fractionated on a methylmercury agarose gel.The RNA contained intact light chain and heavy chain mRNA's as inferredfrom specific hybridization to kappa UIG-MJK primer, and to the mouseheavy chain UIG-MJH3 probe (see FIG. 7). In addition, the UIG-MJK primerwas used for specific priming of anti-HBsAg poly(A)⁺ RNA in a dideoxysequencing reaction. Sufficient sequence was read to show that a majorkappa RNA of the anti-HBsAg cell line contains the J_(K) 2 sequence.

The conditions for variable region cDNA synthesis were optimized byusing heavy and light chain UIG primers on anti-HBsAg poly(A)⁺ RNA.Dideoxy chain extension experiments demonstrated that the mouse UIG-MJKprimer and UIG-JH3 primer correctly primed kappa and heavy chain RNAs.When the reverse transcription was carried out in the absence ofdideoxynucleotides, the main product using the kappa UIG-MJK primer wasa 410±20 nucleotide fragment, while the main product using the heavychain UIG-JH3 primer was a 430±30 nucleotide fragment. These correspondto the expected lengths of the variable and 5' untranslated regions ofkappa and heavy chain immunoglobulin mRNAs. The conditions for theoptimal priming of poly(A)⁺ RNA from CRL8017 cells should work well forpoly(A)⁺ RNA isolated from any cell line producing a monoclonalantibody.

After determining optimal conditions for priming hybridoma mRNA witholigonucleotide primers, two oligonucleotides were designed and used forheavy chain V region cDNA synthesis. These two oligonucleotides areUIG-MJHBSTEII(13) and UIG-MJH3 (FIGS. 7 and 8). It should be noted thatthe primer sequence was designed to introduce a BstEII recognition site(GGTGACC) in the clone so that it could be joined at this site to thehuman IgG1 constant module at the analogous position at the latter's Jregion. In this case, the primer had a single G to U mismatch with themouse mRNA sequence that uses the J_(H) 3 coding sequence. TheUIG-MJHBSTEII(13) primer was 13 bases long and the mismatched residuewas flanked by 7 matches 5' and 5 matches 3' of it. This was the 13-merBstEII primer. To assess the priming efficiency of the 13-mer BstEIIoligonucleotide, a 21-met primer specific for mouse J_(H) 3 (UIG-MJH3)was used. This primer had a perfect match for the 17 nucleotides on its3' end.

These two primers and the J_(H) 3 coding sequences are shown in FIG. 8.The first strand cDNA products made via the 13-met BstEII and the 21-merJ_(H) 3 primers included bands of approximately 430 nucleotides, whichrepresented the entire V_(H) region. Under the standard primingconditions used, the priming efficiency of the 13-met BstEII was muchless than that of the 21-mer J_(H) 3. Accordingly, a cDNA library wasgenerated from the first strand synthesis from each of these primers,using the method of Gubler and Hoffman, supra.

First, the 21-mer J_(H) 3 library was screened with the 21-mer J_(H) 3oligonucleotide. Filter hybridization was done at 30° , overnight,according to de Lange, T. et al., Cell, 34: 891-900 (1983). The filterswere then washed at 51° in 6×SSC, 0.1% SDS. Five colonies were selected.The largest had an insert of approximately 460 bp. More significantly,it contained three restriction sites predicted from the known J_(H) 3sequence, which are present upstream of the primer sequence. This clone,pJ3-11, was sequenced using the J_(H) 3 primer by the chain-terminationmethod (Wallace, R. B. et al., Gene, 16: 21-26 (1981)). The sequenceobtained has the remaining J_(H) 3 coding segment. Just upstream, a13-nucleotide segment matched to a published D segment sequence (Dsp2.2) (Kurosawa, Y. et al., J. Exp. Med., 155: 201 (1982), and Tonegawa,S., Nature, 302: 575 (1983)). A nonapeptide predicted from this areashowed characteristic homology to the published mouse heavy chain Vsubgroups at amino acid residues 86 to 94, comprising the FR3 of heavychain molecules. Plasmid pJ3-11 represented a rearranged VDJ sequence,and apparently contained the anti-hepatitis V_(H) sequence produced bythe cell line.

In order to isolate a V_(H) region cDNA clone that had a BstEII site inthe J region, an AluI to Sau96I, 265 nucleotide long, probe from pJ3-11was next used to screen the cDNA library generated from the 13-merBstEII primer. Six positive clones were isolated. The largest, pBs13-1,was further analyzed. The insert was 280 nucleotides long and itsrestriction map agreed with that of pJ3-11 except for the introducedBstEII site. FIG. 9 illustrates how these two inserts were recombined togenerate pMVHCa-13, a V_(H) clone with the module-joining BstEII site.Three additional V_(H) cDNA clones were isolated from a cDNA librarygenerated from the 21-mer oligonucleotide UIG-MJH3BSTEII primercontaining a BstEII site. These clones may provide alternate V_(H) cDNAsequences to join to human C_(H) sequences.

(2) Preparation of cDNA Clones, and Vehicles Containing Same, for LightChain Mouse Anti-HBsAg Variable Region

Since the J_(K) 2 sequence is present in mRNA prepared from theanti-hepatitis hybridoma cell line, the oligonucleotide UIG-JK2BGLII(FIG. 7B), was designed to introduce a BglII site into the J_(K) 2region. Digestion with BglII would then allow direct insertion of aV_(K) cDNA coding region into the BclI site of the previously notedhuman C_(K) vector, pING2001, This insertion would result in the precisejoining of a mouse variable region segment (including the J region) to ahuman kappa constant region segment, each in the proper coding frame andwith no alteration in amino acid sequence for either mouse variable orhuman constant region.

The JK2BGLII oligonucleotide was used to prime anti-HBsAg mRNA to form acDNA library as for heavy chain, supra, in pUC9. The cDNA wassize-selected by polyacrylamide gel electrophoresis prior to cloning,and 80% of the cDNA clones were shown to have insert sizes between 300and 750 nucleotides in length. Replica filters of this library werescreened with two oligonucleotides, the original primer and a secondprobe complementary to J_(K) 2 sequence 5' to the original primer.

It was discovered that the anti-hepatitis B monoclonal cell line CRL8017 secretes immunoglobulins with at least two different light chains.One of them is derived from the myeloma NS-1, which was used as a fusionpartner in generating the anti-hepatitis B cell line. Since NS-1 isderived from the myeloma MOPC21, the possibility was investigated thatMOPC21 V_(K) mRNA may be present in the V_(K) cDNA library from theanti-hepatitis monoclonal cell line. Indeed, one cDNA clone (p6D4B)analyzed has an identical restriction enzyme map to that of MOPC21 V_(K)cDNA, except for the inserted BglII site.

Two conclusions can be drawn from these results. The first is that it ispossible to effectively use an oligonucleotide to introduce arestriction enzyme site while cloning a V_(K) region from a hybridomacell line. The second is that one must carefully monitor hybridoma celllines for the presence of multiple V region sequences, only one of whichis the desired sequence.

In order to further characterize the kappa light chain J regions presentin the cell line mRNA, poly(A)⁺ RNA was bound to nitrocellulose by theformaldehyde "Dot blot" procedure of White and Bancroft, J. Biol. Chem.,257:8569 (1982). The RNA was hybridized to ³² P-labeled oligonucleotideprobes specific for each functional kappa J region. These probes areshown in FIG. 7B as the UIG probes 5JK1, MJK, 5JK4, and 5JK5. Theresults showed that the mRNA hybridized strongly to both MJK and 5JK4oligonucleotide probes, indicating that both J_(K) 2 and J_(K) 4sequences were present. Since J_(K) 2 mRNA had been previouslyidentified as the one derived from the parental hybridoma partner NS-1,it was concluded that the J_(K) 4 mRNA encoded the anti-hepatitisbinding specificity of the CRL 8017 cells.

Two different cDNA libraries were screened to isolate late V regionclones encoding J_(K) 4 sequences. The first was primed by JK2BGLII,supra. The second was made by using the oligonucleotide primer,JK4BGLII, which is specific for J_(K) 4 mRNA and introduces a BglII siteinto the J region of cloned V regions. The JK4BGLII primer was used toprime first strand cDNA synthesis to construct a cDNA library by thesame method used to construct a JK2BGLII primed cDNA library, exceptthat cDNA was not size selected prior to cloning.

FIG. 7B tabulates the mismatches that each primer has with otherfunctional mouse kappa J region sequences. Note that J_(K) 4 has fivemismatches in 21 nucleotides when compared with the JK2BGLII primer, and3 in 23 with the JK4BGLII primer.

Both libraries were screened for V region clones containing J_(K) 4sequences by hybridizing to an oligonucleotide probe specific for J_(K)4 sequences (5JK4). The results of this screen are shown in Table 1.

                  TABLE 1*                                                        ______________________________________                                                   Probe Specificity                                                  Library   J.sub.K 4       J.sub.K 4                                           ______________________________________                                        JK2BGLII  2% (30/1500)    0.15% (2/1500)                                      JK4BGLII  N/D             3.5% (31/875)                                       ______________________________________                                         *Percentage of clones containing J.sub.K 2 or J.sub.K 4 sequence plus a V     region. The probes used were the oligonucleotide 5JK4 (J.sub.K 4              specificity, FIG. 7) and p6D4B, which contains the NS1 (MOPC21) V region      sequence.                                                                     N/D, not done.                                                           

Several J_(K) 4 V region cDNA clones isolated from both libraries werecharacterized. These clones have identical restriction enzyme maps,including the engineered BglII site resulting from the oligonucleotideprimed cDNA cloning procedure. The restriction map and sequence of oneclone, pV17, show that pV17 contains V region gene sequences.

These results show that the JK2BGLII primer could correctly, althoughinefficiently, prime J_(K) 4 mRNA sequences. Since the JK2BGLII primerhad less mismatches with any other J_(K) region mRNA than with J_(K) 4mRNA (FIG. 7B), it is expected that the other J_(K) mRNAs can be primedat the correct location with better efficiency using the JK2BGLIIprimer. Thus, efficient cDNA cloning of any functional mouse kappa Vregion may be obtained by using a mixture of the JK2BGLII and JK4BGLIIprimers.

The placement of a BglII site into the J region during cDNA cloning ofthe V regions allows joining of the cloned mouse V region gene module tothe human kappa constant region gene module (FIG. 9B).

After the aforementioned experiments were carried out it was found thatthe cDNA clone pV17 lacked a complete 5' coding region. Nucleotidesequencing showed that the A of the initiator codon ATG was not copiedin pV17. This was not a random cDNA cloning artifact because two othercDNA clones had the same defect. Two approaches were devised to obtain alight chain gene with a complete 5' coding region.

First, a new cDNA library was constructed by first priming with anoligonucleotide (5'-ATATTTGCTGATGCT CT-3') complementary to pV17sequences 155 bases from the 5' end. From this library, cloneshybridizing to a pV17 DNA fragment probe were selected, and some ofthese new cDNA clones have the initiator ATG plus about 20 nucleotidesof 5' untranslated region. One of these clones, p2-12, supplies a 5'untranslated region of 23 nucleotides and a complete ATG initiatorcodon. When p2-12 was combined with pV17 derived sequences, a variableregion with a complete 5' end was formed (pING2013E).

Second, site-directed mutagenesis on the existing light chain clone wasused to simultaneously remove the poly-G tract and place a ribosomerecognition sequence adjacent to the initiator ATG. The PstI fragmentfrom pV17 was subcloned into M13mp18. An oligonucleotide (V17-IVM;5'-GTGTCGACTCAGCATGAGGTTCC AGGTTC-3') was then used as a primer tomutate the pV17 sequence to include a SalI site and an initiator ATGinto the pV17 sequence. The resultant plasmid pV17-IVM provided analternate mouse variable-region for joining to human constant regionmodules.

The complete nucleotide sequence of the variable region from pV17 wasthen determined. The sequence shows that pV17 contains a V_(K) -J_(K)junction region, containing several conserved amino acids, and thehybrid J_(K) 2/J_(K) 4 region formed by priming the J_(K) 4 RNA with theUIG-JK2BGLII oligonucleotide. However, the V_(K) region in pV17 isnon-functional, because the V_(K) and J_(K) regions are not in the samecoding frame. Translation of the pV17 V region would thus result in anabnormal immunoglobulin light chain where the J region is translated inan incorrect frame. This defect may be caused by aberrant V-J joining,resulting in a non-functional kappa mRNA, as has been observed byKelley, D. E. et al., Mol. Cell. Biol., 5:1660-1675 (1985).

Since the pV17 V region encodes an abnormal immunoglobulin, it is highlyunlikely that this light chain is part of a functional anti-hepatitisantibody molecule. These results show the importance of monitoringhybridoma cells for the presence of multiple RNA species encoding Vregions, only one of which is the desired sequence.

Further screening of CRL 8017 cDNA libraries was done to search forV_(K) cDNA clones which are not from either of the two V_(K) cDNAclasses found so far (MOPC21-p6D4B, pV17). First an oligo-dT primed cDNAlibrary made from CRL8017 RNA was screened with a DNA fragment probespecific for the kappa constant region, and separately with probesspecific for MOPC21 and pV17 V_(K) regions. A cDNA clone (plE9L-81) thatcontains the kappa constant region, but has a different V_(K) regionthan that of MOPC21 or pV17 was discovered. This method of screeningoligo-dT primed cDNA libraries is a useful alternative tooligonucleotide screening of cDNA libraries, because nick-translatedprobes of high specific activity are used. Also, this method allows thesimultaneous isolation of several classes of V region clones, such asall V_(K) clones, by appropriate probe choice. Second, theUIG-JK2BGLII-primed cDNA library made from CRL 8017 RNA was screenedwith the UIG-5JK2 oligonucleotide probe (see FIG. 7). A new class ofV_(K) cDNA clones was found whose members are homologous,to plE9L-81 andhybridize to the UIG-5JK2 probe, but not to a MOPC21 V_(K) probe. Therestriction endonuclease site maps and nucleotide sequences of theseclones also differ from MOPC21-homologous V_(K) cDNA clones from CRL8017cells. These clones, however, have an aberrant V-J joint which resultsin a nonfunctional mRNA, and appear to be identical to one described byCabilly and Riggs (Gene, 40:157 (1985)).

It was therefore concluded that the anti-hepatitis B cell line CRL8017has at least three classes of V_(K) mRNA corresponding to the abovedescribed cDNA clones p6D4B (MOPC21), p1E9L, and pV17. The pIE9L andpV17 clones are derived from mRNA from aberrantly rearranged Kappagenes, while the p6D4B clone is derived from the parent hybridoma fusionpartner NS-1. None of these clones appear to encode the desiredanti-hepatitis light chain.

(3) Preparation and Expression of Heavy Chain Containing HumanConstant/Mouse Variable Regions

The V region sequences in pMVHCa-13 were joined to the human IgG1constant (C) region clone pGMH-6. Due to the presence of a second BstEIIsite within the IgG1 CHl region of pGMH-6, a multi-step ligation wasrequired. First, the 220 nucleotide BstEII fragment from the J-CHlregion of pGMH-6 was ligated to the 1100 nucleotide IgG region BstEII toBamHI fragment of pGMH-6. In a separate ligation, the 420 nucleotideBstEII to BamHI fragment of pMVHCa-13, which comprises the mouse Vregion, was joined to a calf intestine phosphatase treated BamHI plasmidvector. The two ligations were then combined, ligase was added, and theproducts were transformed into HB101, resulting in the chimeric mouseV-human C clone pMVHCc-24 (FIG. 9A).

The V region of the hybrid heavy chain gene in pMVHCc-24 was furtheranalyzed by partial sequence analysis. This analysis showed that thecloned V region contained a D sequence which matches a known D sequence,DSP2.2 (Kurosawa and Tonegawa, supra). The sequence also predicted a 19amino acid leader peptide similar to known mouse V heavy chain leaderpeptide sequences, and a 5' untranslated region of at least 3nucleotides.

The BamHI fragment containing the mouse-human hybrid heavy chain gene ofpMVHCc-24 was cloned into BamHI digested pING2003E vector, resulting inthe express/on plasmid pING2006E (FIG. 11). The pING2006E plasmid shouldhave an increased probability of efficient expression of the mouse-humanchimeric immunoglobulin gene in B lymphoid cells because of the presenceof the mouse heavy chain enhancer region.

A modification of the chimeric heavy chain gene present in pMVHCc-24 wasdone to provide an alternate heavy chain gene which lacks the oligo-dCregion preceding the initiator ATG. The pING2012E and pING2006E vectorsare identical except for the nucleotides immediately preceding the ATG,as shown in FIG. 12.

Bacteria harboring the pING2006E and pSV2-neo plasmids were convertedinto protoplasts by the method of Sandri-Goldin, R. M. et al., Mol.Cell. Biol., 1: 743 (1981). The protoplasts were then separately fusedto SP2/0-Ag14 hybridoma cells (ATCC CRL 1581) by treatment withpolyethyleneglycol (Ochi, A. et al., Nature, 302: 340, 1983). The fusedcells were allowed to recover for 72 hours in complete medium beforeplating at 10,000 or 50,000 cells per well in a 96-well tissue cultureplate. The cells were selected with G418 at 0.8 mg/ml for two weeks,when growth in some wells was clearly evident. Under these selectionconditions, Sp2/0 cells were completely killed within 4-7 days by G418.Only cells which have integrated and expressed the neo gene present inthe vectors will grow under G418 selection. The number of wells positivefor growth by these integrative transfectants are shown in Table 2.

                  TABLE 2*                                                        ______________________________________                                        Strain/          10,000      50,000                                           Plasmid          cells/well  cells/well                                       ______________________________________                                        MC1061/pING2006E 3 (13%)     12 (50%)                                         MC1061/pSV2-neo  7 (29%)      4 (17%)                                         MC1061/none      0            1                                               ______________________________________                                         *Percentage of wells showing positive growth out of 24 wells.            

Cells transfected with pING2006E and pSV2-neo were tested forimmunoglobulin gene expression at the RNA and protein level. Total cellRNA was prepared from transfected cells, bound to nitrocellulose andhybridized to nick-translated probes specific for the mouse-human hybridheavy chain gene. Two clones were found which have a strong signal,representing expression of the gene at the RNA level. The amount oftotal cellular RNA hybridizing to the mouse-human probe appeared to beapproximately 1/10 the level of heavy chain RNA in the originalhybridoma cells. This probably represented about 1% of the total mRNA ofthe transfected cell.

The transfected mouse cells were also tested for production ofcytoplasmic human heavy chain protein by an ELISA assay. It was foundthat 3 out of 7 pING2006E transfected cell lines produced detectablelevels of human heavy chain protein. The mouse cell transformantproducing the most mouse-human heavy chain protein gave a signal in theELISA assay comparable to that of a 1/100 dilution of a human B cellline producing intact human immunoglobulin IgG1. This modest level ofdetected mouse-human heavy chain protein may be due to several factors,including instability of heavy chains in the absence of light chains inhybridoma cells, or incorrect processing of the chimeric genetranscript.

(4) Gene Amplification of the Integrated Chimeric Gene

Southern blot analysis showed that multiple copies of the pING2006E DNAsequences were integrated in tandem in the mouse genome. Restrictionenzymes ApaI and BglII both cleave pING2006E singly. In thetransformant, 2AE9, a band, from an ApaI or BglII digestion, of theexpected size (8.2 kb) was found to hybridize to the human C gamma 1sequences (data not shown) and a BamHI band of the correct size (1.6 kb)was found to hybridize to the human as well as the 1E9 V_(H) sequences.A gene-copy titration experiment (FIG. 14) indicated that there areabout 5 copies of pING2006E in the 2AE9 genome. That fact that only asingle band was detected in the ApaI or BglII lane indicates that theseindividual copies are in a tandemly arranged array. A set of doubledigestions showed that pING2006E sequences suffered no rearrangement intheir introduction into the mouse DNA (data not shown).

We next transfected the 2AE9 cells with a plasmid that contains adifferent selectable marker, the gpt gene, and selected clones growingout in DMEM-HAT. One clone, 2BH10, has about 38 ng soluble human gamma 1protein per 10⁶ cells. Southern analysis showed that 2BH10 has about 30copies of pING2006E (FIG. 14). They were amplified from the 5 copies in2AE9 without rearrangement of the DNA sequences. (Compare the 2AE9 panelto the 2BH10). S1 data (data not shown) revealed that this increase intemplate led to a higher amount of IgG gene transcripts. We believe thatthese sequences were co-amplified with contiguous cellular sequences asa result of the second selection.

EXAMPLE III A Human-Mouse Chimeric Antibody with Cancer AntigenSpecificity

(1) Antibody L6

L6 monoclonal antibody (MAb) was obtained from a mouse which had beenimmunized with cells from a human lung carcinoma, after which spleencells were hybridized with NS-1 mouse myeloma cells. The antibody bindsto a previously not identified carbohydrate antigen which is expressedin large amounts at the surface of cells from most human carcinomas,including lung carcinomas (adeno, squamous), breast carcinomas, coloncarcinomas and ovarian carcinomas, while the antigen is only present attrace levels in normal cells from the adult host. MAb L6 is an IgG2a andcan mediate antibody dependent cellular cytotoxicity, ADCC, in thepresence of human peripheral blood leukocytes as a source of effectorcells, so as to lyse L6 positive tumor cells, and it can lyse L6positive tumor cells in the presence of human serum as a source ofcomplement; the lysis is detected as the release of ⁵¹ Cr from labelledcells over a 4 hour incubation period. MAb L6 can localize to L6positive tumors xenotransplanted onto nude mice, and it can inhibit theoutgrowth of such tumors. MAb L6 is described in Cancer Res.46:3917-3923, 1986 (on MAb specificity) and in Proc. Natl. Acad. Sci.83:7059-7063, 1986 (on MAb function ).

(2) Identification of J Sequences in the Immunoglobulin mRNA of L6.

Frozen cells were thawed on ice for 10 minutes and then at roomtemperature. The suspension was diluted with 15 ml PBS and the cellswere centrifuged down. They were resuspended, after washes in PBS, in 16ml 3M LiCl, 6M urea and disrupted in a polytron shear. The preparationof mRNA and the selection of the poly(A+) fraction were carried outaccording to Auffray, C. and Rougeon, F., Eur. J. Biochem. 107:303,1980.

The poly (A+) RNA from L6 was hybridized individually with labeled J_(H)1, J_(H) 2, J_(H) 3 and J_(H) 4 oligonucleotides under conditionsdescribed by Nobrega et al. Anal. Biochem 131:141, 1983). The productswere then subjected to electrophoresis in a 1.7% agarose-TBE gel. Thegel was fixed in 10% TCA, blotted dry and exposed for autoradiography.The result showed that the L6 v_(H) contains J_(H) 2 sequences.

For the analysis of the V_(K) mRNA, the dot-blot method of White andBancroft J. Biol. Chem. 257: 8569, (1982) was used. Poly (A+) RNA wasimmobilized on nitrocellulose filters and was hybridized to labeledprobe-oligonucleotides at 40° in 4×SSC. These experiments show that L6contains J_(K) 5 sequences. A faint hybridization to J_(K) 2 wasobserved.

(3) V Region cDNA Clones.

A library primed by oligo (dT) on L6 poly (A+) RNA was screened forkappa clones with a mouse C_(K) region probe. From the L6 library,several clones were isolated. A second screen with a 5' J_(K) 5 specificprobe identified the L6 (J_(K) 5) light-chain clones. Heavy chain clonesof L6 were isolated by screening with the J_(H) 2 oligonucleotide.

The heavy and light chain genes or gene fragments from the cDNA clones,pH3-6a and pL3-12a were inserted into M13 bacteriophage vectors fornucleotide sequence analysis. The complete nucleotide sequences of thevariable region of these clones were determined (FIGS. 15 and 16) by thedideoxy chain termination method. These sequences predict V region aminoacid compositions that agree well with the observed compositions, andpredict peptide sequences which have been verified by direct amino acidsequencing of portions of the V regions.

The nucleotide sequences of the cDNA clones show that they areimmunoglobulin V region clones as they contain amino acid residuesdiagnostic of V domains (Kabat et al., Sequences of Proteins ofImmunological Interest; U.S. Dept of HHS, 1983).

The L6 V_(H) belongs to subgroup II. The cDNA predicts an N-terminalsequence of 24 amino acid residues identical to that of a known V_(H)(45-165 CRI; Margolies et al. Mol. Immunol. 18:1065, 1981). The L6 V_(H)has the J_(H) 2 sequence. The L6 V_(L) is from the V_(K) -KpnI family(Nishi et al. Proc. Nat. Acd. Sci. USA 82:6399, 1985), and uses J_(K) 5.The cloned L6 V_(L) predicts an amino acid sequence which was confirmedby amino acid sequencing of peptides from the L6 light chaincorresponding to residues 18-40 and 80-96.

(4) In Vitro Mutagenesis to Engineer Restriction Enzyme Sites in the JRegion for Joining to a Human C-Module, and to Remove Oligo (dC)Sequences 5' to the V Modules.

Both clones generated from priming with oligo (dT) L6 V_(K) and L6 V_(H)need to be modified. For the L6 V_(K), the J-region mutagenesis primerJ_(K) HindIII, as shown in FIG. 17B, was utilized. A human C_(K) modulederived from a cDNA clone was mutagenized to contain the HindIIIsequence (see FIG. 17A). The mutagenesis reaction was performed on M13subclones of these genes. The frequency of mutant clones ranged from 0.5to 1% of the plaques obtained.

It had been previously observed that the oligo (dC) sequence upstream ofthe AUG codon in a V_(H) chimeric gene interferes with proper splicingin one particular gene construct. It was estimated that perhaps as muchas 70% of the RNA transcripts had undergone the mis-splicing, wherein acryptic 3' splice acceptor in the leader sequence was used. Thereforethe oligo (dC) sequence upstream of the initiator AUG was removed in allof the clones.

In one approach, an oligonucleotide was used which contains a SalIrestriction site to mutagenize the L6 V_(K) clone. The primer used forthis oligonucleotide-directed mutagenesis is a 22-mer which introduces aSalI site between the oligo (dC) and the initiator met codon (FIG. 19).

In a different approach, the nuclease BAL-31 was used to chew away theoligo (dC) in the L6 V_(H) clone pH3-6a. The size of the deletion in twoof the mutants obtained was determined by nucleotide sequencing and isshown in FIG. 17. In both of these mutants (delta 4 and delta 21), allof the oligo (dC) 5' to the coding region were deleted.

These clones were then modified by oligonucleotide-directed mutagenesiswith the MJH2-ApaI primer (FIG. 17). This 31-base primer introduces anApaI site in the mouse C_(H) gene at a position analogous to an existingApaI site in human Cgamma1 cDNA gene module. The primer introduces theappropriate codons for the human C gamma 1 gene. The chimeric heavychain gene made by joining the mutagenized mouse V_(H) gene module to ahuman C_(H) module thus encodes a chimeric protein which contains nohuman amino acids for the entire V_(H) region.

The human C gamma 1 gene module is a cDNA derived from GM2146 cells(Human Genetic Mutant Cell Repository, Newark, N.J.). This C gamma 1gene module was previously combined with a mouse V_(H) gene module toform the chimeric expression plasmid pING2012E.

(5) L6 Chimeric Expression Plasmids.

L6 chimeric heavy chain expression plasmids were derived from thereplacement of the V_(H) module pING2012E with the V_(H) modules ofmutants delta 21 and delta 4 to give the expression plasmids pING2111and pING2112 (FIG. 17). These plasmids direct the synthesis of chimericL6 heavy chain when transfected into mammalian cells.

For the L6 light chain chimeric gene, the SalI to HindIII fragment ofthe mouse V_(K) module was joined to the human C_(K) module by theprocedure outlined in FIG. 18, forming pING2119. Replacement of the neosequence with the E. coli gpt gene derived from pSV2-gpt resulted inpING2120, which expressed L6 chimeric light chain and confersmycophenolic acid resistance when transfected into mammalian cells.

The inclusion of both heavy and light chain chimeric genes in the sameplasmid allows for the introduction into transfected cells of a 1:1 generatio of heavy and light chain genes leading to a balanced gene dosage.This may improve expression and decrease manipulations of transfectedcells for optimal chimeric antibody expression. For this purpose, theDNA fragments derived from the chimeric heavy and light chain genes ofpING2111 and pING2119 were combined into the expression plasmid pING2114(FIG. 19). This expression plasmid contains a selectable neo^(R) markerand separate transcription units for each chimeric gene, each includinga mouse heavy chain enhancer.

The modifications and V-C joint regions of the L6 chimeric genes aresummarized in FIG. 20.

(6) Stable Transfection of Mouse Lymphoid Cells for the Production ofChimeric Antibody.

Electroporation was used (Potter et al. supra; Toneguzzo et al. Mol.Cell Biol. 6:703 1986) for the introduction of L6 chimeric expressionplasmid DNA into mouse Sp2/0 cells. The electroporation technique gave atransfection frequency of 1-10×10⁻⁵ for the Sp2/0 cells.

The two gene expression plasmid pING2114 was linearized by digestionwith AatII restriction endonuclease and transfected into Sp2/0 cells,giving approximately fifty G418 resistant clones which were screened forhuman heavy and light chain synthesis. The levels of chimeric antibodychain synthesis from the two producers, D7 and 3E3, are shown in Table3. Chimeric L6 antibody was prepared by culturing the D7 transfectantcells for 24 hours at 2×10⁶ cells/ml in 5 1 DMEM supplemented with HEPESbuffer and penicillin and streptomycin. The supernatant was concentratedover an Amicon YM30 membrane in 10 mM sodium phosphate buffer, pH8.0.The preparation was loaded over a DEAE-Cellulose column, which separatedthe immunoglobulin into unbound and bound fractions. Samples from theDEAE-unbound, DEAE-bound and the pre-DEAE preparations (from 1.6 ul ofmedium) was separately purified by affinity chromatography on aProtein-A Sepharose column, eluting with 0.1M sodium citrate,

pH 3.5. The eluted antibody was neutralized and concentrated by Amiconcentricon filtration, in phosphate-buffered saline. The yields for thethree preparations were 12 ug (DEAE unbound), 6 ug (DEAE bound), and 9ug (pre-DEAE column). Western analysis of the antibody chains indicatedthat they were combined in an H₂ L₂ tetramer like nativeimmunoglobulins.

(7) A second purification for Chimeric L6 Antibody Secreted in TissueCulture.

a. Sp2/0.pING2114.D7 cells were grown in culture medium [DMEM] (Gibco#320-1965), supplemented with 10% Fetal Bovine Serum (Hyclone#A-1111-D), 10 mM HEPES, 1× Glutamine-Pen-Strep (Irvine Scientific#9316) to 1×10⁶ cell/ml.

b. The cells were then centrifuged at 400×g and resuspended inserum-free culture medium at 2×10⁶ cell/ml for 18-24 hr.

c. The medium was centrifuged at 4000 RPM in a JS-4.2 rotor (3000×g) for15 min.

d. 1.6 liter of supernatant was then filtered through a 0.45 micronfilter and then concentrated over a YM30 (Amicon Corp.) filter to 25 ml.

e. The conductance of the concentrated supernatant was adjusted to5.7-5.6 mS/cm and the pH was adjusted to 8.0.

f. The supernatant was centrifuged at 2000×g, 5 min., and then loadedonto a 40 ml DEAE column, which was preequilibrated with 10 mM sodiumphosphate, pH8.0.

g. The flow through fraction was collected and loaded onto a 1 mlprotein A-Sepharose (Sigma) column preequilibrated with 10 mM sodiumphosphate, pH8.0.

h. The column was washed first with 6 ml 10 mM sodium phosphate bufferpH=8.0, followed by 8 ml 0.1M sodium citrate pH=3.5, then by 6 ml 0.1Mcitric acid (pH=2.2). Fractions of 0.5 ml were collected in tubescontaining 50 ul 2M Tris base (Sigma).

i. The bulk of the IgG was in the pH=3.5 elution and was pooled andconcentrated over Centricon 30 (Amicon Corp.) to approximately 0.06 ml.

j. The buffer was changed to PBS (10 mM sodium phosphate pH=7.4, 0.15MNaCl) in Centricon 30 by repeated diluting with PBS and reconcentrating.

k. The IgG solution was then adjusted to 0.10 ml and bovine serumalbumin (Fraction V, U.S. Biochemicals) was added to 1.0% as astabilizing reagent.

(8) Production and Purification of Chimeric L6 Antibody Secreted inAscites Fluid.

a. The ascites was first centrifuged a6 2,000×g for 10 min.

b. The conductance of the supernatant was adjusted to 5.7-5.6 mS/cm andits pH adjusted to 8.0.

c. Supernatant was then loaded onto a 40 ml DEAE-cellulose columnpre-eguilibrated with 10 mM Na₂ HPO₄.

d. The flow through from the DEAE column was collected and its pH wasadjusted to 7.4, and then loaded onto a 1.0 ml goat anti-human IgG (H+L)-sepharose column.

e. The column was washed first with 6 ml of 10 mM sodium phosphate, 0.5Msodium chloride, followed by 8 ml of 0.5M NH₄ OH, and 3M sodiumthiocyanate.

f. The sodium thiocyanate eluate was pooled and dialyzed against 2L PBSovernight.

The antibody can be further concentrated by steps j. and k. of theprevious procedure.

                  TABLE 3                                                         ______________________________________                                        Levels of Secreted Chimeric L6                                                Chains from Sp2/0 Transfectants.sup.a                                                      Sp2/0.D7   Sp2/0.3E3                                             Culture Condition                                                                        FBS     Kappa.sup.b                                                                           Gamma.sup.c                                                                          Kappa.sup.b                                                                         Gamma.sup.c                           ______________________________________                                        1.   20 ml, 2d,                                                                              +       17    77     100   700                                     seed @                                                                        2 × 10.sup.5 /ml                                                    2.  200 ml, 2d,                                                                              +       0.9   6      80    215                                     seed @                                                                        2.5 × 10.sup.5 /ml                                                  3.  200 ml, 1d,                                                                              -       1.9   3.8    97    221                                     seed @                                                                        2 × 10.sup.6 /ml                                                    4.  Balb/c ascites                                                                           -       5,160 19,170 ND    ND                                  ______________________________________                                         .sup.a -- Sp2/0 cells transfected by electroporation with pING2114 (pL6HL     .sup.b -- ug/l measured by ELISA specific for human Kappa  human              BenceJones protein standard.                                                  .sup.c -- ug/l measured by ELISA specific for human gamma  human IgG          standard.                                                                     ND -- Not determined.                                                         FBS: Fetal Bovine Serum                                                  

(9) Studies Performed on the Chimeric L6 Antibody.

First, the samples were tested with a binding assay, in which cells ofboth an L6 antigen-positive and an L6 antigen-negative cell line wereincubated with standard mouse monoclonal antibody L6, chimeric L6antibody derived from the cell culture supernatants, and chimeric L6antibody derived from ascites (as previously described) followed by asecond reagent, fluo-rescein-isothiocyanate (FITC)-conjugated goatantibodies to human (or mouse, for the standard) immunoglobulin.

Since the binding assay showed strong reactivity of the chimeric L6 onthe L6 antigen positive cell line and total lack of reactivity on thenegative cell line, the next step was to test for the ability of thechimeric L6 to inhibit the binding of mouse L6 to antigen positivecells; such inhibition assays are used routinely to establish theidentity of two antibodies' recognition of antigen. These data arediscussed below ("Inhibition of binding"). As part of these studies, arough estimate of antibody avidity was made.

Finally, two aspects of antibody function were studied, the ability tomediate ADCC in the presence of human peripheral blood leukocytes, andthe ability to kill L6 positive tumor cells in the presence of humanserum as a source of complement (see "Functional Assays" below).

Binding Assays. Cells from a human colon carcinoma line, 3347, which hadbeen previously shown to express approximately 5×10⁵ molecules of the L6antigen at the cell surface, were used as targets. Cells from the T cellline HSB2 was used as a negative control, since they, according toprevious testing, do not express detectable amounts of the L6 antigen.The target cells were first incubated for 30 min at 4° C. with eitherthe chimeric L6 or with mouse L6 standard, which had been purified frommouse ascites. This was followed by incubation with a second,FITC-labelled, reagent, which for the chimeric antibody wasgoat-anti-human immunoglobulin, obtained from TAGO (Burlingame, Calif.),and used at a dilution of 1:50. For the mouse standard, it wasgoat-anti-mouse immunoglobulin, also obtained from TAGO and used at adilution of 1:50. Antibody binding to the cell surface was determinedusing a Coulter Model EPIC-C cell sorter.

As shown in Table 4 and Table 4A, both the chimeric and the mousestandard L6 bound significantly, and to approximately the same extent,to the L6 positive 3347 line. They did not bind above background to theL6 negative HSB2 line.

In view of the fact that the three different chimeric L6 samplespresented in Table 4 behaved similarly in the binding assays, they werepooled for the inhibition studies presented below. The same inhibitionstudies were performed for chimeric L6 derived from ascites fluidpresented in Table 4A.

Inhibition of Binding. As the next step was studied the extent to whichgraded doses of the chimeric L6 antibody, or the standard mouse L6,could inhibit the binding of an FITC-labelled mouse L6 to the surface ofantigen positive 3347 colon carcinoma cells.

Both the chimeric and mouse standard L6 inhibited the binding of thedirectly labelled L6 antibody, with the binding curves being parallel.The chimeric antibody was slightly less effective than the standard, asindicated by the results which showed that 3.4 ug/ml of the pooledchimeric L6 MAb, as compared to 2.0 ug/ml of the standard mouse L6 MAbwas needed for 50% inhibition of the binding, and that 5.5 ug/ml of thechimeric L6 (derived from ascites) as compared to 2.7 ug/ml of thestandard mouse L6 MAb was needed for 50% inhibition of binding.

As part of these studies, a rough estimate was made of antibody avidity.The avidity of the standard mouse L6 had been previously determined tobe approximately 4×10⁸ The data indicated that there were no significantdifferences in avidity between the chimeric and the mouse L6.

Functional Assays. A comparison was made between the ability of thechimeric L6 and standard mouse L6 to lyse L6 antigen positive cells inthe presence of human peripheral blood leukocytes as a source ofeffector cells (mediating Antibody Dependent Cellular Cytotoxcity, ADCC)or human serum as a source of complement (mediating Complement-DependentCytolysis, CDC).

As shown in Table 5 and Tables 5A-5D, the chimeric L6 was superior tothe simultaneously tested sample of mouse L6 in causing ADCC, asmeasured by a 4 hr ⁵¹ Cr release test.

Tables 6 and 6A-6B present the data from studies on complement-mediatedtarget cell lysis. In this case, a high cytolytic activity was observedwith both the mouse and the chimeric L6 antibodies.

Conclusions.

The results presented above demonstrate a number of important,unexpected qualities of the chimeric L6 monoclonal antibody of theinvention. Firstly, the chimeric L6 antibody binds to L6 antigenpositive tumor cells to approximately the same extent as the mouse L6standard and with approximately the same avidity. This is significantfor the following reasons: the L6 antibody defines (a) a surfacecarbohydrate antigen, and (b) a protein antigen of abut 20,000 daltons,each of which is characteristic of non-small cell lung carcinoma (NSCLC)and certain other human carcinomas. Significantly, the L6 antibody doesnot bind detectably to normal cells such as fibroblasts, endothelialcells, or epithelial cells in the major organs. Thus the chimeric L6monoclonal antibody defines an antigen that is specific for carcinomacells and not normal cells.

In addition to the ability of the chimeric L6 monoclonal antibodies ofthe present invention to bind specifically to malignant cells andlocalize tumors, the chimeric L6 exerts profound biological effects uponbinding to its target, which make the chimeric antibody a primecandidate for tumor immunotherapy. The results presented hereindemonstrate that chimeric L6 is capable of binding to tumor cells andupon binding kills the tumor cells, either by ADCC or CDC. Such tumorkilling activity was demonstrated using concentrations of chimeric L6antibody as low as 0.01 ug/ml (10 ng/ml).

Although the prospect of attempting tumor therapy using monoclonalantibodies is attractive, with some partial tumor regressions beingreported, to date such monoclonal antibody therapy has been met withlimited success (Houghton, February 1985, Proc. Natl. Acad. Sci.82:1242-1246). The therapeutic efficacy of mouse monoclonal antibodies(which are the ones that have been tried so far) appears to be too lowfor most practical purposes. The discovery of the profound biologicalactivity of chimeric L6 coupled with its specificity for a carcinomaantigen makes the chimeric L6 antibody a choice therapeutic agent forthe treatment of tumors in vivo. Moreover, because of the "human"properties which will make the chimeric L6 monoclonal antibodies moreresistant to clearance in vivo, the chimeric L6 monoclonal antibodieswill be advantageously used not only for therapy with unmodifiedchimeric antibodies, but also for development of variousimmunoconjugates with drugs, toxins, immunomodulators, isotopes, etc.,as well as for diagnostic purposes such as in vivo imaging of tumorsusing appropriately labelled chimeric L6 antibodies. Suchimmunoconjugation techniques are known to those skilled in the art andcan be used to modify the chimeric L6 antibody molecules of the presentinvention.

Two illustrative cell lines secreting chimeric L6 antibody weredeposited prior to the filing date of this application at the ATCC,Rockville Md. These are transfected hybridoma C255 (corresponds to 3E3cells, supra), ATCC HB 9240 and transfected hybridoma C256 (D7 cells,supra), ATCC HB 9241.

The present invention is not to be limited in scope by the cell linesdeposited since the deposited embodiment is intended as a singleillustration of one aspect of the invention and all cell lines which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those shown in theart from the foregoing description and accompanying drawings areintended to fall within the scope of the appended claims.

                  TABLE 4                                                         ______________________________________                                        Binding Assays Of Chimeric L6 Antibody and Mouse L6                           Monoclonal Antibody on an L6 Antigen Positive and L6                          Antigen Negative Cell Line.                                                   Antibody   Batch       GAM      GAH                                           ______________________________________                                                         Binding Ratio For*                                                            H3347 Cells (L6+)                                            Standard L6            56.6     4.2                                           Chimeric L6                                                                              a           1.3      110.3                                                    b           1.3      110.3                                                    c           1.3      110.3                                                          Binding Ratio For*                                                            HSB-2 Cells (L6-)                                            Standard L6            1.1      1.1                                           Chimeric L6                                                                              a           1.0      1.0                                                      b           1.0      1.1                                                      c           1.0      1.1                                           ______________________________________                                         *All assays were conducted using an antibody concentration of 10 ug/ml.       The binding ratio is the number of times brighter a test sample is than a     control sample treated with GAM (FITC conjugated goatanti-mouse) or GAH       (FITC conjugated goat antihuman) alone. A ratio of 1 means that the test      sample is just as bright as the contol; a ratio of 2 means the test sampl     is twice as bright as the control, etc.                                  

                  TABLE 4A                                                        ______________________________________                                        Binding Assays Of Chimeric L6 Antibody and Mouse Mono-                        clonal Antibody on an L6 Antigen Positive and L6 Antigen                      Negative Cell Line.                                                                     Antibody                                                                      Concentration                                                       Antibody  (ug/ml)       GAM      GAH                                          ______________________________________                                                          Binding Ratio For*                                                            H3347 Cells (L6+)                                           Standard L6                                                                             30            38       4                                                      10            49       4                                                      3             40       3                                            Chimeric L6                                                                             30            2        108                                          (Ascites) 10            2        108                                                    3             1        42                                           Chimeric L6                                                                             30            1        105                                          (Cell Culture)                                                                          10            1        86                                                     3             1        44                                                             Binding Ratio For**                                                           HSB-2 Cells (L6-)                                           Standard L6                                                                             10            1        1                                            Chimeric L6                                                                             10            1        1                                            (Ascites)                                                                     Chimeric L6                                                                             10            1        1                                            (Cell Culture)                                                                ______________________________________                                         *The binding ratio is the number of times brighter a test sample is than      control sample treated with GAM (FITC conjugated goat antihuman) alone. A     ratio of 1 means that the test sample is just as bright as the control; a     ratio of 2 means the test sample is twice as bright as the control, etc. 

                  TABLE 5                                                         ______________________________________                                        ADCC of Chimeric L6 (Mouse) L6 Antibodies On                                  Colon Carcinoma Cell Line 3347.                                                         Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                             10           100        64                                                    5            100        70                                                    10           0          2                                           Standard L6                                                                             10           100        24                                                    5            100        17                                                    10           0          2                                           None      0            100        1                                           ______________________________________                                         *The target cells had been labelled with .sup.51 Cr and were exposed for      hours to a combination of MAb and human peripheral blood leukocytes (PBL)     and the release of .sup.51 Cr was measured subsequently. The release of       .sup.51 Cr (after corrections of values for spontaneous release from          untreated cells) is a measure of the percent cytolsis.                   

                  TABLE 5A                                                        ______________________________________                                        ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On                     Colon Carcinoma Cell Line 3347.                                                         Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                             20           100        80                                          (Ascites) 10           100        74                                                    5            100        71                                                    2.5          100        71                                                    20           0          0                                           Chimeric L6                                                                             10           100        84                                          (Cell Culture)                                                                          5            100        74                                                    2.5          100        67                                                    10           0          3                                           Standard L6                                                                             20           100        32                                                    10           100        26                                                    20           0          0                                           ______________________________________                                         *The target cells had been labelled with .sup.51 Cr and were exposed for      hours to a combination of MAb and human peripheral blood leukocytes (PBL)     and the release of .sup.51 Cr was measured subsequently. The release of       .sup.51 Cr (after corrections of values for spontaneous release from          untreated cells) is a measure of the percent cytolsis.                   

                  TABLE 5B                                                        ______________________________________                                        ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On                     Colon Carcinoma Cell Line 3347.                                                         Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                             5            100        84                                          (Ascites) 2.5          100        78                                                    1.25         100        85                                                    0.63         100        81                                                    0.31         100        80                                                    0.16         100        71                                                    0.08         100        65                                                    5            0          0                                           Standard L6                                                                             5            100        32                                                    5            0          0                                           None      0            100        19                                          ______________________________________                                         *The target cells had been labelled with .sup.51 Cr and were exposed for      hours to a combination of MAb and human peripheral blood leukocytes (PBL)     and the release of .sup.51 Cr was measured subsequently. The release of       .sup.51 Cr (after corrections of values for spontaneous release from          untreated cells) is a measure of the percent cytolsis.                   

                  TABLE 5C                                                        ______________________________________                                        ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On                     Lung Carcinoma Cell Line H2669.                                                         Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolyis*                                   ______________________________________                                        Chimeric L6                                                                             10           100        35                                          (Ascites) 1            100        31                                                    0.1          100        27                                                    0.01         100        15                                                    0.001        100        13                                                    0.0001       0          15                                          Standard L6                                                                             10           100        9                                                     1            100        15                                          None      0            100        9                                           Chimeric L-6                                                                            10           10         19                                          (Ascites) 1            10         15                                                    0.1          10         11                                                    0.01         10         13                                                    0.001        10         22                                                    0.0001       10         11                                          Standard L6                                                                             10           10         7                                                     1            10         6                                           None      0            10         8                                           Chimeric L6                                                                             10           0          4                                           (Ascites)                                                                     Standard L6                                                                             10           0          9                                           ______________________________________                                         *The target cells had been labelled with .sup.51 Cr and were exposed for      hours to a combination of MAb and Human peripheral blood leukocytes (PBL)     and the release of .sup.51 Cr was measured subsequently. The release of       .sup.51 Cr (after corrections of values for spontaneous release from          untreated cells) is a measure of the percent cytolysis.                  

                  TABLE 5D                                                        ______________________________________                                        ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On                     Colon Carcinoma Cell Line H3347.                                                        Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                             10           100        62                                          (Ascites) 1            100        66                                                    0.1          100        69                                                    0.01         100        26                                                    0.001        100        8                                                     0.0001       0          3                                                     10           0          0                                           Standard L6                                                                             10           100        19                                                    1            100        24                                                                 0          0                                           None      0            100        8                                           ______________________________________                                         *The target cells had been labelled with .sup.51 Cr and were exposed for      hours to a combination of MAb and Human peripheral blood leukocytes (PBL)     and the release of .sup.51 Cr (after corrections of values for spontaneou     release from untreated cells) is a measure of the percent cytolysis.     

                  TABLE 6                                                         ______________________________________                                        Complement-dependent cytotoxic effect of chimeric and                         standard (mouse) L6 on colon carcinoma cells from line 3347,                  as measured by a 4-hr .sup.51 Cr-release assay. Human serum                   from a healthy subject was used as the source of complement.                  Antibody      Human complement                                                                             % Ctyolysis                                      ______________________________________                                        L6 Standard 10 ug/ml                                                                        Yes            90                                               L6 chimeric 10 ug/ml                                                                        Yes            89                                               L6 Standard 10 ug/ml                                                                        No             0                                                L6 chimeric 10 ug/ml                                                                        No             1                                                ______________________________________                                    

                  TABLE 6A                                                        ______________________________________                                        Complement Dependent Cytotoxic Effect of Chimeric L6                          and Standard (Mouse) L6 Antibodies on Colon Carcinoma                         Cell Line 3347                                                                           Antibody                                                                      Concentration                                                                             PBL per    %                                           Antibody   (ug/ml)     Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                              20          +          29                                          (Ascites)  10          +          23                                                     5           +          18                                                     2.5         +          8                                                      20          Inactivated                                                                              0                                                      10          0          0                                           Chimeric L6                                                                              20          +          29                                          (Cell Culture))                                                                          5           +          26                                                     2.5         +          18                                                     20          +          4                                                      10          0          4                                           Standard L6                                                                              20          +          55                                                     10          +          37                                                     20          Inactivated                                                                              0                                                      20          0          1                                           None       0           +          0                                           ______________________________________                                         *Complement mediated cytolysis was measured by a 4 hour .sup.51 Crrelease     assay. Human serum from a healthy subject was used as the source of           complement.                                                              

                  TABLE 6B                                                        ______________________________________                                        Complement Dependent Cytotoxic Effect of Chimeric L6 and                      Standard (Mouse) L6 Antibodies on Colon Carcinoma                             Cell Line 3347                                                                          Antibody                                                                      Concentration                                                                              PBL per    %                                           Antibody  (ug/ml)      Target Cell                                                                              Cytolysis*                                  ______________________________________                                        Chimeric L6                                                                             10           +          209                                         (Ascites) 5            +          155                                                   2.5          +          166                                                   1.25         +          114                                                   0.6          +          63                                                    0.3          +          17                                                    10           0          0                                           Standard L6                                                                             10           +          96                                                    5            +          83                                                    2.5          +          48                                                    1.25         +          18                                                    0.6          +          7                                                     0.3          +          4                                                     10           0          2                                           None      0            +          0                                           ______________________________________                                         *Complement mediated cytolysis was measured by a 4 hour .sup.51 Crrelease     assay. Human serum from a healthy subject was used as the source of           complement.                                                              

EXAMPLE IV A Human-Mouse Chimeric Antibody with Specificity for HumanB-Cell Antigen

The 2H7 mouse monoclonal antibody (gamma _(2b) K) recognizes a humanB-cell surface antigen, Bp35 (Clark, E. A., et al., Proc. Nat. Acad.Sci. USA 82:1766 (1985)). The Bp35 molecule plays a role in B-cellactivation. mRNA was prepared from the 2H7 cell line. Two cDNA librarieswere generated--one using the heavy chain UIG-H primer and the other,oligo(dT). One V_(H) clone, pH2-11, was isolated upon screening with thesame UIG-H oligonucleotide. To isolate the light chain clone, a mousekappa-specific DNA fragment was used to screen the oligo(dT) library.Candidate clones were further screened with a mouse J_(K) 5 sequences.One V_(K) clone, pL2-12, was thus isolated. The light chain UIG-K wasthen used to engineer a restriction enzyme site in the J region.

The two cDNA clones were also modified at the 5' end to remove theartificial oligo d[C] sequence. In pH2-11 this was carried out by usingthe restriction enzyme NcoI which cuts one nucleotide residue 5' of theATG initiator codon. In pL2-12 this was achieved by an oligonucleotidein vitro mutagenesis using a 22-mer container a SalI site.

The DNA sequences of these two clones are shown in FIGS. 21, 22. Toconstruct the chimeric heavy chain plasmid, the V_(H) module was joinedto the human C gamma 1 module (pGMH6) at the J_(H) BstEII site, and thechimeric light chain the V_(K) module was joined to the human C_(K)module (pGML60) at the J_(K) HindIII site. The expression vectorsequences were derived from pING2012-neo as well as pING2016-gpt. Theconstructed plasmids are pING2101 (V_(H) C gamma 1-neo), pING2106 (V_(K)C_(K) -neo), pING2107 (V_(K) C_(K) -gpt). pING2101 and pING2106 werealso used to generate plasmids containing both genes. They are pHL2-11and pHL2-26. In addition, pING2106 and pING2014 were combined to a twolight chain plasmid, pLL2-25, to compensate for the poorer (compared toheavy chain) steady-state accumulation of light chain protein intransfected cells. (See FIG. 23.) FIG. 24 shows the changes made to thevariable region sequences during the construction.

The plasmid, pHL2-11, was linearized by AatII; and the DNA was used totransfect Sp2/0 cells by electroporation. Transformants were selected inG418DMEM. One transformant, 1C9, produces 9.3 ng/ml chimeric kappa and33-72 ng/ml chimeric gamma 1 protein as assayed by ELISA. Southernanalysis of 1C9 DNA showed that there is one copy of the plasmidintegrated in the Sp2/0 genome.

EXAMPLE V Secretion of a Functional Chimeric Antibody from Yeast

(1) Fusion of mature chimeric L6 light chain and heavy chain genes tothe yeast invertase signal sequence and shortened phosphoglyceratekinase (PGK promoter).

Yeast cells are capable of recognizing mammalian secretion signalsequences and of directing secretion of mammalian proteins (Hitzman etal., supra). There is, however, evidence which suggests that certainnative yeast signal sequences are more effective than mammalian signalsequences at directing secretion of some mammalian proteins from yeast(Smith et al., Science 292:1219 (1985)). one example is the signalsequence for the yeast invertase gene. To improve the efficiency oflight and heavy chain secretion, the mature light chain and heavy chainsequences were fused to the yeast invertase signal sequence and placedunder transcriptional control of the shortened PGK promoter (U.S. patentapplication Ser. No. 797,477) using the strategies outlined in FIGS. 25and 26, respectively. An important element of these constructions is theuse of in vitro mutagenesis to introduce a restriction site at thesignal sequence processing site for both the invertase signal sequence(see U.S. patent application Ser. No. 797,477) and the light and heavychain genes. These restriction sites are positioned such that ablunt-ended ligation of restriction enzyme-digested, T-4 DNApolymerase-treated DNA results in in-phase translational fusions of the5' end of the mature immunoglobulin chains with the 3' end of the yeastinvertase signal sequence. Such genes, when expressed in a yeast cell,may direct the synthesis, processing, and secretion of chimeric lightand heavy chains with the same primary peptide sequence as chimericlight and heavy chains secreted from transfected mouse Sp2/0 cells. TheDNA sequences of the mutagenesis primers used for light and heavy chaingenes as well as the corresponding unmutagenized sequences are shown inFIGS. 25B and 26B, respectively. Using this approach, the L6 chimericlight and heavy chains were fused to the yeast invertase signal sequenceand shortened PGK promoter, resulting in plasmids pING1407-7 andpING1415 (FIGS. 25C and 26C).

(2) Removal of non-yeast 3' untranslated DNA.

Recent studies on expression of hepatitis B surface antigen in yeastdemonstrated that removal of non-yeast 3' and 5' untranslated sequencescan result in increased levels of heterologous gene expression in yeast(Knieskin et al., Gene 46:135 (1986)). The light chain gene sequence ofchimeric L6 antibody in pING1407-7 (FIG. 25C) contains approximately 200bp of 3' untranslated DNA followed by 70 bp of poly A and 20 bp of polyG sequences. An initial treatment of the chimeric L6 light chain DNAwith the double-stranded exonuclease Bal31, removed the poly A and polyG sequences and all but 90 bp of 3' untranslated DNA, generating theplasmid pING2121b (FIG. 27). A restriction fragment from pING2121bcontaining only C_(k) was cloned into a derivative of pBR322, generatingpING1419 (FIG. 27). A second Bal31 digestion was next used to remove allbut 13 bp of non-yeast 3' untranslated DNA generating the plasmid,pING1431 (FIG. 27). The chimeric L6 heavy chain gene in pING1415 (FIG.26) also contains extensive 3' untranslated sequence which includes 80bp of poly A. All but 11 bp of the 3' untranslated DNA were removedusing the strategy shown in FIG. 28, generating the plasmid pING1429.

Site-directed in vitro mutagenesis can introduce, at a low frequency,unwanted base pair changes in regions of the DNA outside of the areabeing mutagenized. To ensure that such mutations were not present in thechimeric L6 light and heavy chain sequences which had been cloned intoM13 and subjected to site-directed mutagenesis, we constructed light andheavy chain genes fused to the invertase signal sequence and theshortened PGK promoter which consisted of coding sequences that wereeither confirmed by DNA sequence analysis or proven to be functional byvirtue of their expression in transfected mouse Sp2/0 cells to producefunctional chimeric L6 antibody. The plasmids, pING1439 (light chain,FIG. 27) and pING1436 (heavy chain, FIG. 28) were generated by theseconstructions.

(3) Construction of yeast expression plasmids containing chimeric L6light and heavy chain genes from pING1439 and pING1436, respectively,fused to the PGK polyadenylation signal.

In order for yeast to produce an intact functional antibody molecule, abalanced synthesis of both light and heavy chain protein within the hostcell is preferred. One approach is to place the light and heavy chaingenes on separate expression vectors each containing a differentselective marker. A yeast strain defective in the selective markersfound on the plasmids can then be either simultaneously or sequentiallytransformed with these plasmids.

The chimeric L6 light and heavy chain genes from pING1439 (FIG. 27) andpING1436 (FIG. 28) were cloned as BglII-XhoI and BamHI-XhoI fragments,respectively, in two different medium copy number (about 20 copies/cell)expression vectors (yeast-E. coli shuttle). One of these, pING804CVS,contains the complete yeast 2-micron circle, the PGK transcriptiontermination and polyadenylation signals, and the leu2 gene as theselective marker. The other vector, pING1150, contains the yeast originof replication, oriY, a cis-acting sequence (REP3) from the yeastendogenous 2-micron plasmid, the PGK transcription termination andpolyadenylation signals, and the ura3 gene as the selective marker. Bothplasmids also contain the β-lactamase gene (bla) for ampicillinresistance and the bacterial origin of replication (oriB) from pBR322for selection and amplification in bacteria. Four plasmids resulted fromthese constructions: pING144 1--light chain, leu2 and pING1443--lightchain, ura3 (FIG. 29); pING1440--heavy chain, leu2 and pING1442--heavychain, ura3 (FIG. 30).

(4) Secretion of chimeric L6 antibody from transformed yeast cells.

Two separate transformation experiments were performed in an attempt toobtain both light and heavy chain synthesis in yeast cells. Four μg eachof pING1440 and pING1443, and separately of pING1442 and pING1441 werecotransformed into Saccharomyces cerevisiae strains BB331C (MATa, ura3,leu2) by selecting for growth on SD agar (2% glucose, 0.67%yeast-nitrogen base, 2% agar). Ura⁺ Leu⁺ transformants appeared at 2-3days of incubation at 30° C. Approximately 100 transformants wereobtained for pING1440 plus pING1443; only 15 transformants were obtainedfor pING1442 plus pING1441. Ten colonies were inoculated from each plateinto 5 ml SD broth supplemented with 50 mM sodium succinate, pH 5.5, andgrown for 65 hours at 30° C. The cells were removed by centrifugationand the culture supernatants analyzed by ELISA for the levels of lightchain and heavy chain and for the degree of association of the secretedlight and heavy chains. The latter was assessed using a goat anti-humankappa antiserum to coat the microtiter wells and a peroxidase-labeledgoat anti-human gamma antiserum to detect the level of heavy chain boundto the anti-kappa coat. The results of these assays (Table 7) revealedthat all of the culture supernatants from the cells transformed withpING1440 (heavy chain, leu2) plus pING1443 (light chain, ura3) containeda disproportionately high level of light chain protein relative to thelevels of heavy chain protein, and no evidence (at least as determinedby ELISA) of assembled light and heavy chains. On the other hand, thesupernatants from the cells transformed with pING1442 (heavy chain,ura3)+ pING1441 (light chain, leu2) contained a more balanced productionof light and heavy chain proteins, and eight of ten isolates appeared tocontain some assembled light and heavy chains as determined by ELISA.Two of these isolates, No. 1 and No. 5, produced a significantproportion of assembled light and heavy chain.

                  TABLE 7                                                         ______________________________________                                        LEVELS OF SECRETED CHIMERIC L6 LIGHT AND                                      HEAVY CHAIN BY YEAST TRANSFORMANTS.sup.a                                                Isolate                  Kappa/                                     Plasmids.sup.b                                                                          No.     Kappa.sup.c                                                                             Gamma.sup.d                                                                          Gamma.sup.e                                ______________________________________                                        pING1440 +                                                                              1       284       39     0                                          pING1443  2       324       33     0                                                    3       473       52     0                                                    4       387       40     0                                                    5       316       34     0                                                    6       188       28     0                                                    7       381       45     0                                                    8       455       45     0                                                    9       380       26     0                                                    10      579       32     0                                          pING1441 +                                                                              1       128       79     35                                         pING1442  2       150       30     1                                                    3       124       29     0                                                    4       185       55     5                                                    5       114       52     35                                                   6       139       23     5                                                    7       149       34     5                                                    8       245       57     12                                                   9       202       26     11                                                   10      157       19     7                                          ______________________________________                                         .sup.a. S. cerevisiae strain BB331C (MATa, leu2, ura3) transformed to         Ura.sup.+ Leu.sup.+ with plasmids carrying ura3 and leu2 with light or        heavy chains.                                                                 .sup.b. Plasmids:                                                             pING1440 = heavy chain + leu2;                                                pING1443 = light chain + ura3;                                                pING1442 = heavy chain + ura3;                                                pING1441 = light chain + leu2.                                                .sup.c. ng/ml measured by ELISA specific for human kappa with human Bence     Jones protein as standard.                                                    .sup.d. ng/ml measured by ELISA specific for human gamma with human as Ig     standard.                                                                     .sup.e. ng/ml measured by ELISA using antihuman kappa as coating antibody     and antihuman gamma as second antibody with human IgG standard.          

Further analysis was performed to determine if this association was theresult of the synthesis of an H₂ L₂ -size protein. The culturesupernatants from isolates Nos. 1 and 5, as well as from isolate No. 8,which contained a much lower level of apparent light and heavy chainassociation, were concentrated by ultra-filtration on a Centricon 30filter (Amicon Corp.). The concentrated supernatants were run on a 7%polyacrylamide gel under non-reducing conditions, blotted tonitrocellulose, and probed with goat anti-human kappa antiserum followedby peroxidase-labeled rabbit anti-goat antiserum. The concentratedsupernatants from isolates No. 1 and 5, but not from No. 8, contained asingle immunoreactive band which co-migrated with the purified chimericL6 antibody from transfected Sp2/0 cells. These results suggested thatisolates No. 1 and 5 were synthesizing and secreting assembled L6chimeric antibody.

(5) Purification of chimeric L6 antibody from yeast culture supernatant.

In order to further characterize the H₂ L₂ -size protein secreted by theyeast and determine if this was assembled L6 chimeric antibody, asufficient quantity of yeast-produced material was purified to allow theperformance of various binding and functional assays. The pING1442+1441transformant isolate No. 5 was grown for 58 hours at 30° C. in a10-liter fermentor using a synthetic medium (Table 8). The cells wereinitially grown in 9 liters of the column A medium until the glucoselevel fell below 1 g/L at which time they were fed with a total volumeof 2.5 L of medium from column B. Glucose levels were maintained at 0.5g/L during the remaining course of the fermentation. The cells wereremoved by centrifugation and the culture supernatant was analyzed byELISA for the presence of light and heavy chain proteins and forassociation of the heavy and light chains. The supernatant containedapproximately 250 μg/L of light chain, 240 μg/L of heavy chain, and 130μg/L of heavy chain associated with light chain. The culturesupernatants were next concentrated by ultrafiltration over a D.C. 10unit (Amicon Corp.), filtered through 0.45 micron filter andconcentrated over a YM30 filter (Amicon Corp.) to 250 ml. Theconcentrated supernatant was adjusted to pH 7.4 with KOH, brought to 500ml with PBS (10 mM sodium phosphate, pH 7.4, 150 mM sodium chloride) andloaded on a 1 ml protein A-Sepharose (Sigma) column, pre-equilibratedwith PBS. The column was washed first with 20 ml PBS, followed by 10 ml0.1M sodium citrate, pH 3.5, then by 10 ml 0.1M citric acid pH=2.2. ThepH 3.5 and 2.2 eluates were each collected in a tube containing 1 ml 2MTris base (Sigma). The bulk of the light and heavy chain immunoreactiveproteins were in the pH 3.5 eluate which was next concentrated over aCentricon 30 (Amicon Corp.) to a final volume of 106 ul. Analysis ofthis protein on non-reducing polyacrylamide gels using coomassie bluestaining and immunoblotting with anti-human kappa antiserum (Sigma) tovisualize the proteins revealed an H₂ L₂ -size, 150 kilodaltons, proteinband. This protein was purified away from other proteins by HPLC usingan AB_(x) 5-micron column equilibrated with buffer A (10 mM KPO₄, pH6.8). After loading the sample on the column, the column was washed withbuffer A for 10 minutes (flow rate=1 ml/minute) and subjected to alinear gradient of 0% to 50% buffer B (250 mM KPO₄, pH 6.8) over 50minutes at 1 ml/minute.

                  TABLE 8                                                         ______________________________________                                        MEDIUM USED FOR YEAST FERMENTATION TO                                         PRODUCE SECRETED L6 CHIMERIC ANTIBODY.sup.a                                   Ingredients      A.sup.b         B.sup.c                                      ______________________________________                                         1. Cerelose (Glucose)                                                                         119     g/l     538  g/l                                      2. (NH.sub.4).sub.2 SO.sub.4                                                                  13.9    g/l     83.3 g/l                                      3. Thiamine HCL 0.011   g/l     0.05 g/l                                      4. Biotin       0.00011 g/l     0.005                                                                              g/l                                      5. Pantothenic acid                                                                           0.002   g/l     0.009                                                                              g/l                                      6. Inositol     0.194   g/l     0.875                                                                              g/l                                      7. H.sub.3 PO.sub.4                                                                           5.67    ml/l    25.5 ml/l                                     8. KH.sub.2 PO.sub.4                                                                          5.78    g/l     26.0 g/l                                      9. MgSO.sub.4.7H.sub.2 O                                                                      3.33    g/l     15.2 g/l                                     10. CaCl.sub.2.2H.sub.2 O                                                                      0.33    g/l     1.5  g/l                                     11. FeSO.sub.4.7H.sub.2 O                                                                      0.072   g/l     0.34 g/l                                     12. ZnSO.sub.4.7H.sub.2 O                                                                      0.022   g/l     0.104                                                                              g/l                                     13. MnCl.sub.2.4H.sub.2 O                                                                      0.0039  g/l     0.018                                                                              g/l                                     14. CuSO.sub.4.5H.sub.2 O                                                                      0.0067  g/l     0.031                                                                              g/l                                     15. Conc.H.sub.2 SO.sub.4                                                                      0.0056  ml/l    0.026                                                                              ml/l                                    ______________________________________                                         .sup.a. Fermentation was performed as described in text.                      .sup.b. Constituents of initial 9liter batch.                                 .sup.c. Constituents of 2.5 liter feed batch.                            

The bulk of the protein resolved into a single large broad peak between20 and 50 minutes as determined by absorbance at 280 nm. A secondsmaller peak was observed at 52-56 minutes, which corresponded to thenormal elution position for chimeric L6 antibody from transfected Sp2/Ocells. ELISA analysis of the column fractions revealed a majorheavy+light chain cross-reactive peak corresponding to the U.V.absorbance peak at 52-56 minutes. Analysis of the 52-56 minute fractionson non-reducing SDS polyacrylamide gels using coomassie blue stainingand immunoblotting revealed an essentially pure protein whichco-migrated with L6 chimeric antibody purified from transfected Sp2/Ocells.

(6) Studies performed on the chimeric L6 antibody secreted by yeast.

The purified yeast-derived antibody was assessed for function in severalways. First, the purified antibody was tested for its ability to binddirectly to an L6 antigen-positive cell line. Second, the antibody wastested for its ability to inhibit binding of mouse L6 antibody toantigen-positive cells. Finally, the purified antibody was tested fortwo aspects of antibody function--the ability to mediate ADCC in thepresence of human peripheral blood leukocytes and the ability to kill L6positive tumor cells in the presence of human complement.

Direct Binding Assay. Cells from a human colon carcinoma line, 3347,which expresses approximately 5×10⁵ molecules of the L6 antigen per cellon the cell surface, were used as targets. Cells from the T cell line,T51, were used as a negative control since they, according to previoustesting, do not express detectable amounts of the L6 antigen. The targetcells were first incubated for 30 min at 4° C. with either the Sp2/Ocell- or yeast-derived chimeric L6 antibody or with mouse L6 antibodystandard purified from mouse ascites. This was followed by incubationwith FITC-labeled goat-anti-human immunoglobulin for the chimericantibodies or with FITC-labeled goat-anti-mouse immunoglobulin for themouse standard. Both labeled antibodies were obtained from TAGO(Burlingame, Calif.) and used at a dilution of 1:50. Antibody binding tothe cell surface was determined using a Coulter Model EPIC-C cellsorter.

As shown in Table 9, both the mammalian and yeast-derived chimeric L6antibodies bound significantly, and to approximately the same extent, tothe L6 positive 3347 line. They did not bind above background to the L6negative T51 line.

Inhibition of Binding. As the next step, the yeast chimeric L6 antibodyand the Sp2/O cell-derived chimeric L6 antibody were tested for theirability to inhibit the binding of an FITC-labeled mouse L6 antibody tothe surface of antigen-positive 3347 colon carcinoma cells.

Both the yeast-derived and Sp2/O-derived chimeric L6 antibodiesinhibited the binding of labeled mouse L6 antibody and the bindingcurves were parallel. Based on the results of these studies, a roughestimate was made of antibody avidity. The avidity of the Sp2/Ocell-derived chimeric L6 had been previously determined to beapproximately 4×10⁸. The data indicated that there were no significantdifferences between the avidities of yeast-derived chimeric L6 and Sp2/Ocell-derived chimeric L6 antibodies for the L6 antigen.

Functional Assays. A comparison was made between the ability of theyeast-derived chimeric L6, Sp2/O cell-derived chimeric L6 and standardmouse L6 antibodies to lyse L6 antigen-positive cells in the presence ofhuman peripheral blood leukocytes as a source of effector cellsmediating Antibody Dependent Cellular Cytotoxicity (ADCC). As shown inTable 10, the chimeric L6 from yeast was slightly better thanSp2/O-cell-derived chimeric L6 and as previously observed, both weresuperior to the standard mouse L6 in causing ADCC, as measured by afour-hour ⁵¹ Cr release test.

A comparison was next made between the yeast-derived chimeric L6, Sp2/Ocell-derived chimeric L6 and standard mouse L6 antibodies for theirabilities to lyse L6 antigen-positive cells by complement-dependentcytolysis (CDC) when human serum was used as the source of complement.The results of this comparison (Table 11) demonstrated that while boththe Sp2/O-cell-derived chimeric L6 and standard mouse L6 antibodiesexhibited high cytolytic activity, the yeast-derived L6 antibody failedto cause any cytolysis even at the highest antibody concentration. Theseresults were unexpected and demonstrate that the yeast-derived antibodyhas new and unique properties.

(7) Conclusions

A process is disclosed by which yeast can be genetically engineered tosecrete functional anti-bodies. The yeast-derived chimeric antibody inthis example binds to the appropriate target antigen with approximatelythe same avidity as the chimeric antibody produced by lymphoid (Sp2/0)cells. The yeast-derived antibody also displays similar ADCC activity asdoes Sp2/0-derived antibody. Unlike the Sp2/0 cell-derived antibody, theyeast-derived antibody displayed no CDC activity, thus demonstrating thenew and unique properties of the yeast-derived antibody. This processshould be applicable for the production of a variety of monoclonalanti-bodies and chimeric anti-bodies carrying chosen antigen bindingdomains linked to a chosen constant domain isotype. Geneticallyengineered anti-bodies and derivatives thereof produced in yeast alsowill exhibit novel functional properties, for example, the ability toselectively mediate target killing by ADCC without any detectable CDCactivity. The technology described herein may also be suitable for theproduction of various other heterologous multimeric secreted proteins bygenetically engineered yeast.

                  TABLE 9                                                         ______________________________________                                        BINDING ASSAYS OF CHIMERIC L6 ANTIBODY                                        PRODUCED BY YEAST OR MOUSE Sp2/O CELLS ON AN                                  L6 ANTIGEN-POSITIVE AND AN L6 ANTIGEN-                                        NEGATIVE CELL LINE                                                                          Binding Ratio.sup.b for:                                                        H3347 Cells                                                                              T51 Cells                                          Antibody.sup.a  (L6+)      (L6-)                                              ______________________________________                                        Standard Mouse L6                                                                             95         1.0                                                Sp2/O Chimeric L6                                                                             116        1.0                                                Yeast Chimeric L6                                                                             116        1.0                                                ______________________________________                                         .sup.a. All antibodies were used at a concentration of 10 μg/ml.           .sup.b. The binding ratio is the number of times brighter a test sample i     than a control sample treated with FITCconjugated second antibody. Goat       antimouse antibody was used as the second antibody for standard mouse L6      monoclonal antibody. Goat antihuman antibody was used as the second           antibody for the yeast and Sp2/O chimeric L6 antibody.                   

                  TABLE 10                                                        ______________________________________                                        ADCC OF CHIMERIC L6 ANTIBODY DERIVED FROM                                     YEAST OR Sp2/O CELLS AND STANDARD (MOUSE) L6                                  ANTIBODY ON COLON CARCINOMA CELL LINE 3347                                                    Antibody                                                                      Concentration                                                                             %                                                 Antibody        (μg/ml)  Cytolysis*                                        ______________________________________                                        Standard mouse L6                                                                             5.0         42                                                                1.0         48                                                Sp2/O Chimeric L6                                                                             1.0         96                                                                0.1         71                                                                0.01        54                                                                0.001       37                                                Yeast Chimeric L6                                                                             1.0         114                                                               0.1         108                                                               0.01        76                                                                0.001       60                                                None            0           23                                                ______________________________________                                         *The target cells had been labeled with .sup.51 Cr and were exposed for       four hours to a combination of MAb and human peripheral blood leukocytes      at 100 per target cell, and the release of .sup.51 Cr was measured            subsequently. The release of .sup.51 Cr (after corrections of values for      spontaneous release from untreated cells) is a measure of the percent         cytolysis.                                                               

                  TABLE 11                                                        ______________________________________                                        HUMAN COMPLEMENT-DEPENDENT CYTOTOXIC                                          EFFECTS OF CHIMERIC L6 ANTIBODY PRODUCED                                      BY YEAST OR MOUSE Sp2/O CELLS                                                 ON COLON CARCINOMA CELL LINE 3347                                                         Antibody                                                                      Concentration                                                                             Complement.sup.a                                                                          Percent                                   Antibody    (μg/ml)  (+ or -)    Cytolysis                                 ______________________________________                                        Standard mouse L6                                                                         5           +           122                                                   1           +           53                                                    5           -           1                                         Sp2/O Chimeric L6                                                                         5           +           73                                                    1           +           22                                                    0.1         +           5                                                     5           -           2                                         Yeast Chimeric L6                                                                         5           +           3                                                     1           +           2                                                     0.1         +           4                                                     5           -           2                                         ______________________________________                                         .sup.a Human serum from a healthy subject was used as the source of           complement.                                                                   .sup.b Complementmediated cytolysis was measured by a fourhour .sup.51        Crrelease assay.                                                         

EXAMPLE VI Secretion of Functional Chimeric Fab from Yeast

The Fab portion of IgG consists of a single light chain molecule coupledby a disulfide bridge to a single truncated heavy chain moleculeconsisting of the variable region and C_(H) 1 (FIG. 31). This heavychain fragment is known as Fd. Fabs are potentially useful for a varietyof therapeutic and diagnostic procedures. In addition, they are amenableto production by microbial fermentation.

The usual method for production of Fab involves the digestion of intactIgG with papain (see FIG. 31) followed by purification of the Fab awayfrom the Fc fragments generated in the digest. While this procedure isrelatively straightforward and can result in high yields of Fab, it issomewhat time-consuming in that it first requires the production andpurification of whole antibody followed by generation and, finally,purification of Fab. Furthermore, one-third of the whole antibodymolecule--the Fc portion (FIG. 31)--is not utilized.

The recent advances in gene cloning and site-specific mutagenesistechnology make possible a more direct and simple alternative approachfor production of Fab molecules. In this approach, a stop codon isintroduced in the heavy chain gene within the hinge region atapproximately the codon for the amino acid at which papain digestionoccurs. The Fab is then produced directly by simultaneous expression ofboth the light chain and Fd genes to produce their respective proteinswhich assemble and are secreted from the cell.

(1) Introduction of a stop codon in the hinge region of L6 chimericheavy chain.

The strategy for introduction of a stop codon into the hinge region ofL6 chimeric heavy chain is outlined in FIG. 32A. The location of thestop codon within the hinge region and the DNA sequence of themutagenesis primer are shown in FIG. 32B. The stop codon placementcorresponds to amino acid 226 in FIG. 31. This procedure generated theplasmid pING1402 containing an Fd gene which codes for a proteinconsisting of 228 amino acids and extends six amino acids beyond thecysteine to which the light chain is coupled. The mutagenesis alsointroduced a unique BclI site at the stop codon which can be readilyutilized for subsequent manipulations of the 3' end of Fd. Theseinclude, but are not necessarily limited to, removal of heavy chain 3'untranslated DNA as well as the engineering of various types ofmodifications of Fd including the addition of coding sequences forspecific amino acids and the production of fusion proteins.

(2) Fusion of the mature Fd gene to yeast invertase signal sequence andshortened PGK promoter.

The strategy for fusion of the Fd gene to the yeast invertase signalsequence is outlined in FIG. 33. This approach made use of the priorconstruction of the yeast invertase signal sequence--mature L6 heavychain fusion (FIG. 26) and utilized a unique ApaI site in the J regionof the chimeric L6 heavy chain to replace the constant region inpING1415 consisting of C_(H) 1, C_(H) 2, and C_(H) 3 with the constantregion from pING1412 containing the stop codon in the hinge region. Thisprocedure generated the plasmid, pING1418.

(3) Removal of non-yeast 3' untranslated DNA.

The introduction of a unique BclI site at the stop codon of the Fd chainprovided a convenient method for removal of all non-yeast 3'untranslated DNA. This was accomplished using the strategy outlined inFIG. 34, and generated the plasmid, pING1428.

Since the stop codon was introduced into the hinge region bysite-specific mutagenesis of a heavy chain fragment cloned into M13, thepossibility existed that unwanted mutations could have been introducedduring the mutagenesis step. To ensure that such mutations were notpresent, an Fd gene fused to the invertase signal sequence and shortenedPGK promoter and consisting of known coding sequences was constructedusing the strategy outlined in FIG. 34, generating the plasmid,pING1444.

(4) Construction of yeast expression plasmids containing the chimeric L6Fd gene from pING1444 fused to the PGK polyadenylation signal.

In order for yeast to produce an intact, functional Fab molecule, abalanced synthesis of both light and Fd-chain proteins must occursimultaneously within the cell. As described in Example V, one approachis to place the light chain and Fd genes on separate shuttle vectorscontaining separate selective markers and to transform these vectorsinto a yeast strain defective for both selective markers.

The Fd gene from pING1444 (FIG. 34.) was cloned as a BamHI-XhoI fragmentinto two medium copy number yeast-E. coli shuttle vectors containingsequences for replication in yeast and the PGK polyadenylation,transcription termination signal: pING804CVS for led2 selection andpING1150 for ura3 selection (see FIGS. 29, 30). The two plasmidsresulting from these constructions--pING1445 (ura3) and pING1446 (leu2)are shown in FIG. 35.

(5) Secretion of chimeric L6 Fab from transformed yeast cells.

Two separate transformation experiments were performed in an attempt toobtain both light and Fd-chain synthesis in yeast cells. Four μg each ofpING1445 (FIG. 35) and pING1441 (FIG. 30) and separately of pING1446(FIG. 35) and pING1442 (FIG. 30) were co-transformed into S. cerevisiaestrain BB331c (MATa, ura3, leu2) by selecting for growth on SD agar (2%glucose, 0.67% yeast nitrogen base, 2% agar). Ura⁺ Leu⁺ transformantsappeared at two to three days of incubation at 30° C.

Five colonies were inoculated from each plate into 6 ml SD brothsupplemented with 50 mM sodium succinate, pH 5.5, and grown for 65 hoursat 30° C. The cells were removed by centrifugation and analyzed by ELISAfor the levels of light chain. The results of these assays revealed thatthe levels of light chain in the culture supernatants of the pING1446+pING1443 transformants were three to six times higher than the levels inthe culture supernatants of the pING1445+ pING144 1 transformants. Theculture supernatants for each group of transformants were nextconcentrated by ultrafiltration on a Centricon 30 filter (Amicon Corp.)and run on a 10% polyacrylamide gel under non-reducing conditions. Theproteins were blotted to nitrocellulose paper and probed with goatanti-human kappa antiserum followed by peroxidase-labeledrabbit-anti-goat antiserum. The concentrated supernatant from thepING1446 and pING1443 transformants contained a significant anti-kappacross-reactive smear over a large portion of the blot with only a faintcross-reactive band at the J position expected for the Fab protein. Bycomparison, the concentrated supernatants from pING1445+ pING144 1transformants contained relatively little smeared anti-human kappacross-reactive protein on the blot. In addition, one of the five samples(No. 4) contained an especially intense, distinct anti-kappacross-reactive band which migrated at the position expected for an Fabprotein.

(6) Purification of chimeric L6 Fab from yeast culture supernatant.

To establish that the Fab-size anti-kappa cross-reactive proteinsecreted by the yeast is indeed L6 chimeric Fab protein required thepurification of sufficient quantities for performance of binding assays.The pING1441+ pING1445 transformant isolate No. 4 was, therefore, grownin one liter of SD broth supplemented with 50 mM sodium succinate, pH5.5, for 95 hours at 30° C. The cells were removed by centrifugation andthe culture supernatant was analyzed by ELISA for the level of lightchain protein. The supernatant contained approximately 130 μg/L of lightchain protein. The culture supernatant was next concentrated byultrafiltration over an Amicon YM30 filter to 20 ml. The concentratedsupernatant was washed with 130 ml 10 mM potassium phosphate, pH 7.5(buffer A) and re-concentrated over the YM30 filter to 12.5 ml. Theconcentrated supernatant was next brought to 54 ml with buffer A andloaded onto a 1.5 ml S-Sepharose column equilibrated with buffer A. Thecolumn was washed with 20 ml buffer A and subjected to a linear gradientof 0 to 200 mM-sodium chloride in buffer A (40 ml total volume). ELISAanalysis of the column fractions revealed a large anti-kappacross-reactive peak between fractions 8 and 21 corresponding to a saltconcentration of approximately 60 mM. These fractions were pooled,concentrated on Amicon YM10 and Centricon-10 filters (Amicon Corp.) to51 μl and analyzed on non-reducing and reducing polyacrylamide gelsusing coomassie blue staining and Western blotting with anti-human kappaand anti-human Fab antisera. These analyses revealed an essentially pureprotein which migrated at approximately 46 kd on the non-reducing geland resolved into two bands running at approximately 23 and 24.5 kd onthe reducing gel which corresponds to the predicted (based on amino acidsequence) molecular weights for light chain and Fd proteins,respectively. The smaller of the two bands strongly reacted withanti-human kappa antiserum on the Western blot. Both of the proteinbands reacted with anti-human Fab antiserum on the Western blot.

(7) Studies performed on the chimeric L6 Fab secreted by yeast.

The primary activity of an Fab molecule is its ability to bind to thetarget antigen. The yeast-derived chimeric Fab was, therefore, testedfor its ability to bind directly to an L6 antigen-positive cell line andfor its ability to inhibit binding of mouse L6 antibody toantigen-positive cells.

Direct Binding Assay. Cells from the human colon carcinoma cell line3347, which contains the L6 antigen at the cell surface, were used astargets. Cells from the antigen-negative cell line, T51, were used as anegative control. The target cells were first incubated for 30 minutesat 4° C. with either yeast-derived chimeric L6 Fab, Sp2/O cell-derivedchimeric L6 antibody, or with mouse L6 antibody. This was followed byincubation with FITC-labelled goat anti-human kappa immunoglobulin forthe chimeric Fab, FITC-labelled goat anti-human IgG for chimericantibody, or with FITC-labelled goat anti-mouse immunoglobulin for themouse antibody. Both labelled anti-bodies were obtained from TAGO(Burlingame, Calif.) and used at a dilution of 1:50. Antibody binding tothe cell surface was determined using a Coulter Model EPIC-C cellsorter.

As shown in Table 12, the yeast-derived chimeric L6 Fab bound to the L6positive 3347 line. The yeast-derived chimeric L6 Fab did not bind abovebackground to the L6 negative T51 line.

Inhibition of Binding. As the next step, we studied the extent to whichgraded doses of the yeast-derived chimeric L6 Fab or Sp2/O-cell-derivedchimeric L6 antibody could inhibit binding of an FITC-labelled mouse L6antibody to the surface of antigen positive colon carcinoma 3347 cells.

The yeast-derived chimeric L6 Fab inhibited the binding of the directlylabeled mouse L6 antibody. A higher concentration of the yeast L6 Fab,however, was required to achieve 50% inhibition of mouse L6 antibodybinding to the target cells than was required for the same degree ofbinding inhibition by Sp2/O cell-derived chimeric L6 antibody.

(8) Conclusions

A process is disclosed by which yeast can be genetically engineered tosecrete functional Fab domains of immunoglobulins. The yeast-derivedchimeric Fab in this example binds to the appropriate target antigen.Such Fab molecules provide convenient targeting agents for a variety ofdiagnostic and therapeutic uses. This process also demonstrates thefeasibility of secretion of heterologous heterodimeric molecules fromyeast.

                  TABLE 12                                                        ______________________________________                                        BINDING ASSAYS OF CHIMERIC L6 FAB PRODUCED                                    BY YEAST ON AN L6 ANTIGEN-POSITIVE AND AND                                    L6 ANTIGEN-NEGATIVE CELL LINE                                                                 Binding Ratio.sup.b for:                                                        3347 Cells                                                                              T51 Cells                                         Antibody.sup.a    (L6+)     (L6-)                                             ______________________________________                                        Sp2/O Chimeric L6 103       1                                                 Yeast Chimeric L6 Fab                                                                           32        1                                                 ______________________________________                                         .sup.a. All antibodies were used at a concentration of 10 μg/ml.           .sup.b. The binding ratio is the number of times brighter a test sample i     than a control sample treated with FITCconjugated second antibody. Goat       antihuman antibody was used as the second antibody for the Sp2/O chimeric     L6 antibody and goatanti-human kappa antibody was used as the second          antibody for the yeast Fab.                                              

EXAMPLE VII Secretion of Functional Chimeric Fab Molecules From Bacteria

Bacteria are suited for production of chimeric anti-bodies expressedfrom mammalian cDNA since entire coding sequences can be expressed fromwell characterized promoters. Escherichia coli is one of many usefulbacterial species for production of foreign proteins (Holland, I. B., etal., BioTechnology 4:427 (1986)), since a wealth of genetic informationis available for optimization of its gene expression. E. coli can beused for production of foreign proteins internally or for secretion ofproteins out of the cytoplasm, where they most often accumulate in theperiplasmic space (Gray et al., Gene 39:247 (1985); Oka et al., Proc.Natl. Acad. Sci. USA 82:7212 (1985)). Secretion from the E. colicytoplasm has been observed for many proteins and requires a signalsequence. Proteins produced internally in bacteria are often not foldedproperly and precipitate into subcellular particles called inclusionbodies (Schoner et al., BioTechnology 3:151 (1985)). Protein secretedfrom bacteria, however, is often folded properly and assumes nativesecondary and tertiary structures (Hsiung et al., BioTechnology 4:991(1986)). Although immunoglobulin peptides have been synthesized ingenetically engineered E. coli (Cabilly et al., Proc. Natl. Acad. Sci.USA 81:3273 (1984); Liu et al., Proc. Natl. Acad. Sci. USA 81:5369(1984); Boss et al., Nucl. Acids Res. 12:3791 (1984)), there are noreports of secretion of these peptides from E. coli as functionalanti-bodies or antibody fragments.

An Fab molecule consists of two nonidentical protein chains linked by asingle disulfide bridge. These two chains are the intact antibody lightchain and the V, J, and C_(H) 1 portions of the antibody heavy chain,Fd. The proper cDNA clones for the L6 chimeric light and Fd gene havealready been identified. In this example, these cDNA clones wereorganized into a single bacterial operon (a dicistronic message) as genefusions to the pectate lyase (pelB) gene leader sequence from Erwiniacarotovora (Lei et al., J. Bacteriol., in press (1987)) and expressedfrom either of two strong, regulated promoters. The result is a systemfor the simultaneous expression of two protein chains in E. coli, andthe secretion of immunologically active, properly assembled Fab of L6chimeric antibody into the culture growth media.

A. Construction of E. coli expression systems for L6 Chimeric Fab

1. Assembly of the pelB leader sequence cassette.

Erwinia carotovora EC codes for several pectate lyases (polygalacturonicacid trans-eliminase) (Lei et al., Gene 35:63 (1985)). Three pectatelyase genes have been cloned, and the DNA sequence of these genes hasbeen determined. When cloned into E. coli under the control of a strongpromoter, the pelB gene is expressed and large quantities of pectatelyase accumulate in the periplasmic space. The pelB signal sequencefunctions efficiently in E. coli and was used as a secretion signal forantibody genes in this example. The nucleotide sequence surrounding thesignal sequence of the pelB gene is shown in FIG. 36a.

The pelB signal sequence contains a HaeIII restriction site at aminoacid 22, adjacent to the signal peptidase cleavage site: ala-ala.Plasmid pSS1004 (Lei et al., J. Bacteriol., in press (1987)), containingthe pelB gene in plasmid vector pUC8 (Vieirra and Messing, Gene 19:259(1982)), was digested with HaeIII and EcoRI. This DNA was ligated withan eight base pair SstI linker to SspI and EcoRI cut pBR322. Theresulting plasmid contained a 300 bp fragment which included the 22amino acid leader sequence of pelB and about 230 bp of upstream E.caratovora DNA. This plasmid. pING173, contains an insert that upondigestion with Sstl and treatment with T4 DNA polymerase can be ligateddirectly to a DNA fragment flanked by the first amino acid of a maturecoding sequence for any gene to generate a protein fusion containing afunctional bacterial leader sequence in frame with the incoming gene.The Sstl to EcoRI restriction fragment in pING173 was cloned into pUC18(Yanich-Perron et al., Gene 33:103 (1985)) to generate pRR175, whichcontains the pelB leader and adjacent upstream non-coding sequence(including a ribosome binding site) downstream of the lac promoter. Theconstruction of pRR175 is outlined in FIG. 36b.

2. Preparation of chimeric L6 light gene for bacterial expression.

The intact L6 chimeric light chain gene containing an AatII restrictionsite at the signal sequence processing site and a unique BglII sitedownstream of the gene was excised from the yeast expression plasmidpING1298 (FIG. 25a) as a 1200 bp DNA fragment. This fragment wasinserted into plasmid pRR175. The resulting plasmid, pRR177-8, containedan in-frame fusion of the pelB leader and the L6 light chain downstreamof the lac promoter residing in the parent plasmid. A number ofderivatives of this plasmid were constructed to delete noncodingsequences from both the 5' and 3' ends of the pelB::light chain genefusion in pRR177-8. Upstream noncoding sequences were deleted making useof an NdeI restriction site at -48 bp from the pelB leader sequenceinitiation codon (FIG. 36) generating pRR180-2. The 3' noncodingsequences were eliminated by substituting a fragment from the plasmidoptimized for L6 light chain expression in yeast, pING1431 (see FIG.27a), into pRR179 to generate pRR191. Another plasmid, pRR190, issimilar to pRR191 but contains 90 bp of noncoding eukaryotic DNA at the3' end of the light chain gene. These constructions are shown in FIG.37.

3. Preparation of chimeric L6 Fd gene for bacterial expression.

The intact L6 chimeric Fd gene containing an SstI restriction site atthe signal sequence processing site, a BclI site introduced by sitedirected mutagenesis (FIG. 32a, b) and creating a termination codon atamino acid 226, and a unique BamHI restriction site downstream of thegene was excised from the plasmid pING1406 (FIG. 33) as a 880 bp DNAfragment. This DNA fragment was inserted into plasmid pRR175 generatingan in-frame fusion of the pelB leader sequence and the L6 Fd genedownstream of the lac promoter, pRR178-5. A number of derivatives wereconstructed to delete noncoding sequences from both the 5' and 3' endsof the sequence contained in pRR178-5. The 3' noncoding sequences wereeliminated by substituting a restriction fragment from the plasmidoptimized for L6 Fd expression in yeast, pING1428 (FIG. 34), whichcontains an XhoI linker immediately following the termination codon ofthe Fd gene, generating plasmid pRR186. Removal of E. caratovora DNAsequences upstream of the NdeI site at -48 from the leader sequencegenerated plasmid pRR196. The construction of these plasmids is shown inFIG. 38.

4. Multicistronic expression system for light chain and Fd gene.

For production of bacterially derived Fab, both light chain and Fd needto be produced simultaneously within the cell. Using the plasmidsconstructed with each of these genes separately, a series of expressionvectors were constructed that contain both genes aligned so thattranscription from a single promoter will specify both genes. This wasdone in a way that minimized the noncoding DNA between the two genes to60 bp. Each gene has a ribosome binding site needed for translationinitiation and the identical DNA sequence from -48 to the pelBleader::antibody gene junction. Several cloning steps were required toalign the two genes together. A portion of the light chain gene linkedto the pelB leader in pRR180-2 was cloned downstream of the Fd gene inpRR186 to generate pFK100. The remainder of the light chain gene wassubcloned into pFK100 from pRR177-8 to generate pFK101. Similarly, DNAfragments containing 3' deletions of eukaryotic sequences from pRR190and pRR191 were cloned into pFK101 generating pFK103 and pFK102respectively. DNA fragments from pRR192 and pFK101 were ligated togenerate pFK104 which contains a deletion of sequences upstream of -48bp from the Fd gene. Maps of the Fd and light chain gene cassettes inthese plasmids are shown in FIG. 39.

5. Placement of the dicistronic message for light chain and Fd under thecontrol of inducible promoters.

Plasmids pFK101, pFK102, pFK103, and pFK104 contain Fd and light chaingenes cloned sequentially under the control of the lac promoter invector pUC18 or pUC19. In E. coli strains such as JM103 F'laciQ (Messinget al., Nucl. Acids. Res. 2:309 (1981)), the amount of light chain thataccumulates in the periplasm is not affected by the lac promoterinducing agent isopropl B-D-thiogalactopyranoside (IPTG), see Table 13.In addition, bacterial growth is slower (compared to cells containingpUC18), and bacterial colonies exhibit an altered morphology beingsmall, dry and rough, suggesting that constitutive foreign geneexpression is deleterious to cell growth. Two strategies were used toplace this gene cassette under more tightly regulated promoters.

First, a PstI to EcoRI fragment from pFK104 was ligated to pIT206 toplace the Fd and light chain gene cassette under the direct control ofthe Salmonella typhimurium araB promoter, a well characterized, strongpromoter in E. coli. A restriction map of pIT206 and construction ofpIT104 is shown in FIG. 40. Use of the araB promoter and its regulatoryprotein araC for the expression of bacterial genes is described in U.S.patent applications Ser. No. 695,309 filed Jan. 28, 1985, and 797,472,filed Nov. 13, 1985. As is seen in Table 14, the resulting plasmid,pIT104, is now regulated for the synthesis of light chain by theaddition of arabinose to the culture growth media. At least 10 foldinduction is effected by arabinose addition. Although Fab secreted intothe growth medium increases more than 10 fold, cell growth stops afterinduction with arabinose. This confirms that high level expression ofthe Fab genes is deleterious to cell growth. Bacterial coloniesharboring pIT104 are phenotypically indistinguishable from E. coliharboring pIT206 when grown in the absence of arabinose.

Second, a DNA fragment containing the laci gene, a repressor of the lacpromoter, was cloned into the high copy expression vector pFK102.Expression of laci from a high copy number vector is useful to regulateexpression of the lac promoter on a high copy number vector (Russel etal., Plasmid, in press (1987); Hsuing et al., Biotechnology 4:991(1986)). A 1.7 kb EcoRI fragment containing the laci gene on pMC9 (Caloset al., Proc. Natl. Acad. Sci. USA 80:3015 (1983)) was excised, filledin with T4 polymerase to blunt ends, ligated with PstI linkers andcloned into the unique PstI site of pFK102 to generate pFK1021laci. Themap of pFK1021laci is shown in FIG. 40b. The selection procedure used toidentify the correct clone assured that the resulting plasmid,pFK1021laci, contained a functionally repressed lac promoter. All whiteor light pink colonies on MacConkey-lactose plates contained plasmidswith laci inserts while transformants containing pFK102 alone were red,indicating the functional repression of the lac promoter by the highcopy number laci gene. Table 14 shows that expression of bacterial Fabfrom cells containing pFK1021laci is similar to expression from pFK102.Unlike cells containing pFK102, which formed aberrant colonies and grewslowly in broth culture, cells containing pFK102laci resembled thosecontaining pUC18.

B. Expression, SDS-PAGE, and Purification of Bacterially Produced Fab

1. Growth of E. coli harboring cloned antibody genes.

Plasmid DNA was transformed into either E. coli JM103 or MC1061 bystandard E. coli transformation procedures. Bacterial cultures Heregrown in TYE (tryptone 1.5%, yeast extract 1.0%, and NaCl 0.5%)supplemented with the appropriate antibiotics (penicillin 250 ug/ml,tetracycline 15 ug/ml). Bacterial cultures were grown in volumes of 5 mlto 1 liter at 37° C. to an optical density OD600=0.8 (approximately4×108 cell/ml) and aliquots were induced with IPTG (0.2 mM), lactose(1.0%), or arabinose (1.0%). Cultures were grown for an additional timeperiod of 4 to 21 hr. Portions of each culture were analyzed for lightchain production. Protein was released from the periplasmic space of E.coli cells by osmotic shock as described (Yanagida et al., J, Bacteriol.166:937 (1986)). Alternatively, culture supernatants were assayeddirectly for the presence of antibody chains.

Quantitation of L6 light chain was by ELISA with goat anti-human Kappalight chain antibody (Cappel, Malvern, Pa.). Fd could be detected byELISA with mouse monoclonal anti- human Fd antibody (Calbiochem, SanDiego, Calif.). Table 13 shows representative data for expression oflight chain reactive material in E. coli periplasmic extracts. Lightchain is secreted from the bacterial cytoplasm into the periplasm.Antibody chains are also released from the bacteria into the culturesupernatant. This is an unusual discovery and may be a unique propertyof the L6 Fab among eukaryotic proteins expressed in E. coli. Undercertain conditions, however, bacterial proteins are known to be releasedfrom E. coli (Abrahmsen et al., Nucl. Acids Res. 14:7487 (1986); Pageset al., J. Bacteriol. 169:1386 (1986)). Table 14 compares the amount oflight chain secreted into the periplasm with the amount secreted intothe culture supernatant. Light chain reactive material is present inplasmid containing cultures harboring cloned light chain alone or lightchain plus Fd. The best producers of Fab (pFK102, pFK104, andpFK102laci) typically secrete 300 -1000 ng/ml of ELISA reactive lightchain into the culture media. A separate construct was made in which thelight chain gene is followed by the Fd gene (pFK107). This constructdirects synthesis and secretion of Fab at similar levels to theconstructs with the genes in the inverse order. Thus, the gene order isnot critical for secretion of Fab.

2. SDS-PAGE of bacterially produced chimeric L6 light chain and Fd.

Bacterially produced antibody chains were analyzed by polyacrylamide gelelectrophoresis under reducing and non-reducing conditions. Proteinextracts of lysed whole bacterial cells, protein released from theperiplasmic space by osmotic shock, and protein secreted into theculture supernatant were analyzed electrophoretically. Transfer of gelseparated protein under full reducing conditions to nitrocellulose andimmunological staining with goat anti-human light chain antibody byWestern analysis revealed that a protein of the same molecular weight asauthentic L6 chimeric light chain was present (about 23 Kd). Analysis ofprotein samples by SDS-PAGE under non-reducing conditions showed thatextracts from cells harboring a plasmid with the light chain gene alone(pRR191 or pRR190) contained a large proportion of the light chainreactive material associated into a higher molecular weight form. Muchof this material ran at about 46 Kd in what is likely to be a lightchain dimer. Light chain dimers have been observed from myeloma cellsproducing only light chain. There are also other immunoreactive proteinbands that may represent non-specific disulfide formation between lightchain and E. coli proteins. Protein samples (periplasmic extracts orculture supernatants) from E. coli cells harboring both the light chainand the Fd genes contain a light chain reactive band at about 48 Kd whenseparated under non-reducing gel conditions which runs at a slightlyhigher molecular weight than the bacterial light chain dimer. Thismaterial is bacterially produced L6 chimeric Fab. In E. coli harboringpFK102laci, pFK101, pFK102, pFK103, or pFK104 the 48 Kd band observed onan SDS gel run under non-reducing conditions is the most prominentimmunoreactive specie. In addition, the background smear ofimmunoreactive proteins seen in extracts containing the light chain onlyis greatly reduced in extracts from cells containing both light chainand Fd.

3. Purification of bacterially produced chimeric L6 Fab.

Immunologically and functionally active (see below) bacterial Fab waspurified from either culture supernatants or periplasmic proteinextracts of E. coli harboring pFK1021aci or pIT104. For purification ofperiplasmic material, the periplasmic fraction from 1 liter of cellsinduced for 4 hours was released into 50 ml of distilled water. Thismaterial was centrifuged for 20 minutes at 5000 g and filtered through a0.45 μm filter. The periplasmic extract was then concentrated over aYM10 membrane (Amicon) to about 5 ml. This material was diluted 8 foldinto starting buffer (10 mM K2HPO4, pH 7.5) and applied to a 1 mlS-Sepharose column at a flow rate of 1.0 ml/min. The column was washedwith 25 ml of starting buffer and eluted with a 0 to 200 mM NaClgradient in starting buffer (200 ml total volume). The immunoreactivegradient peak was pooled (elution was at about 100 mM) and concentratedon a Centricon 10. Purified Fab was stored in PBS+2.0% BSA.

For purification of secreted Fab from 1 liter of bacterial culturesupernatant, the cells were removed by centrifugation after growth for21 hours with inducing agents and the supernatant was filtered through a0.45 μm filter. The media was concentrated over a YM10 membrane (Amicon)to about 16 ml, then diluted with 10 mM K2HPO4 to 105 ml. This materialwas applied to a 1.6 ml S-Sepharose column and eluted with a 0 to 200 mMNaCl gradient in 40 ml. Fab recovered from S-Sepharose chromatographywas greater than 70% pure as determined by densitometry tracing of anonreducing, coomassie stained, 10% acrylamide gel. The Fab purifiedfrom bacterial culture supernatants resolves into two major proteinbands of about 23 Kd and 24.5 Kd on a 15% reducing gel. The molecularweight of Fd and light chain based on the DNA sequence are 24.5 Kd and23 Kd which corresponds well to the observed protein sizes. The smallerof the two bands strongly reacted with goat anti-human Kappa light chainantiserum on a Western blot. Bacterial Fab purified from either theperiplasmic space or bacterial culture supernatants areindistinguishable by all analytical criteria tested here.

4. Functional binding activity of bacterially produced chimeric L6 Fabto the L6 antigen.

Bacterially produced Fab purified by S-Sepharose chromatography wastested for binding to L6 antigen containing cells. As shown in Table 15,bacterial Fab binds specifically to the human colon carcinoma cell line3347. Cells from the T cell line T51 were used as a negative control.Target cells were incubated for 30 minutes at 4° C. with bacteriallyproduced L6 chimeric Fab, intact L6 chimeric antibody produced in Sp2/0cells, or mouse L6 antibody purified from mouse ascites. This wasfollowed by incubation with FITC-labelled goat anti-human light chainantibody for Fab detection, FITC-labelled goat anti-human immunoglobulinfor chimeric antibody detection, or with FITC-labelled goat anti-murineimmunoglobulin for mouse antibody detection. Antibody binding to thecell surface was determined using a Coulter Model EPIC-C cell sorter.

Bacterially produced Fab also exhibits characteristic binding inhibitionof FITC-labelled mouse L6 antibody to the surface of antigen positive3347 colon carcinoma cells. Bacterially produced Fab and Sp2/0 derivedchimeric L6 have similar binding inhibition profiles, thereby suggestingthat the avidity of bacterially produced Fab and Sp2/0 derived chimericL6 are similar.

Conclusions

A novel process is disclosed whereby E. coli has been used as a host toproduce functionally active Fab domains of immunoglobulins and tosecrete these into the periplasmic space and also in the culture medium.This molecule exhibits binding properties expected of a properlyassembled antibody recognition site. This technology can be used toexpress antibody genes with other binding specificities in E. coli.

1. Proteins encoded by modified antibody cDNA clones can be secretedfrom bacteria using a signal sequence.

2. Two antibody genes can be expressed from a single bacterial promoteras a dicistronic message.

3. Two foreign proteins (in this example antibody light chain and Fd)can assemble properly, i.e., assume correct secondary, tertiary, andquaternary structure when secreted from bacteria.

4. At least two, and probably many bacterial promoters can be used forexpression of antibody genes.

5. This example is a general method whereby genes encoding otherantibody chains can be expressed together as a dicistronic message;these include either light chain and Fd genes or light chain and intactheavy chain genes.

6. The gene order with respect to the promoter is not important in theability of E. coli to produce Fab. A construct of the Fd gene followedby the light chain works as well as the genes organized in the inverseorder.

7. Fab can be secreted from E. coli into the culture supernatant whereit is stable and can be purified. Most Fab chains that pass thecytoplasmic membrane are secreted into the culture supernatant.

Microorganism Deposits

Saccharomyces cerevisiae BB331C (41/42-5), G187 was deposited at theATCC on Jul. 9, 1987 and given access number 20856. Escherichia coli JM103 (pFK1021 aci), G186 was also deposited therein on the same date andgiven access number 67457. Both deposits were under the Budapest Treaty.

                  TABLE 13                                                        ______________________________________                                        QUANTITATION OF LIGHT CHAIN FROM                                              E. COLI PERIPLASM                                                             ng/ml of culture           ng/ml of culture                                   plasmid -        +        plasmid                                                                              -      +                                     ______________________________________                                        pRR175  0        0        pFK101 36     28                                    pRR177-8                                                                              8.5      11       pFK102 68     55                                    pRR180  399      412      pFK103 38     45                                    pRR190  200      241      pFK104 91     68                                    pRR191  463      772                                                          ______________________________________                                         E. coli JM103 or MC1061 (results similar) was transformed with each           plasmid. Fresh transformants were cultured in TYE at 37° C. to an      OD600 = 0.8. Cultures were divided and the inducer (IPTG) was added to 0.     mM to one aliquot (- or + IPTG). Cells were grown at 37° C. for 4      hours. Periplasmic protein extracts were prepared, and aliquots were          tested for light chain by ELISA with goat anti human Kappa antibody. Each     value is the average of at least two separate experiments. Removal of         noncoding sequences both 5' and 3' to the antibody gene effected an           increase in light chain accumulation in the periplasm.                   

                  TABLE 14                                                        ______________________________________                                        ACCUMULATION OF LIGHT CHAIN IN THE                                            SUPERNATANT AND PERIPLASM AFTER INDUCTION                                                  Supernatant    Periplasm                                         Plasmid Inducer    4 hr   21 hr   4 hr 21 hr                                  ______________________________________                                        pRR190  -          0      nd      200  nd                                     pRR190  +          5      188     241  nd                                     pFK102  -          12     nd      68   nd                                     pFK102  +          57     828     55   40                                     pFK104  -          13     nd      91   nd                                     pFK104  +          150    290     68   35                                     pFK102laci                                                                            -          25     360     50   100                                    pFK102laci                                                                            +          72     606     37   40                                     pIT104  -          13     nd      10   nd                                     pIT104  +          150    216     19   35                                     ______________________________________                                         Plasmid containing E. coli strains were grown, prepared, and assayed as       described in Table 13. For pRR190, pFK102, pFK104, and pFK102lacicells        were induced with 0.2 mM IPTG; pIT104 was induced with 1% arabinose. Each     value is the average of at least two separate experiments. For analysis o     E. coli culture supernatants, bacteria were removed by centrifugation and     culture supernatants were passed through a 0.45 uM filter. Values are         expressed in ng/ml of culture.                                                nd -- not determined                                                     

                  TABLE 15                                                        ______________________________________                                        BINDING ASSAYS OF BACTERIAL Fab                                                               Binding ratio*                                                                  3347 cells                                                                             T51 cells                                          Antibody          L6+      L6-                                                ______________________________________                                        Standard mouse L6 95       1                                                  Sp2/0 chimeric L6 116      1                                                  Bacterial L6 Fab  54       1                                                  Standard L6 Fab   16       1                                                  ______________________________________                                         *The binding ratio is the number of times brighter a test sample is than      control sample treated with FITCconjugated second antibody.                   Standard L6 Fab was prepared by enzymatic digestion of mouse L6 antibody.

Example VIII EXPRESSION OF PECTATE LYASE B PLASMID CONSTRUCTION

Plasmid pSH2111 (Lei et al., Gene 35: 63-70, 1985) contains the pectatelyase genes from Erwinia carotovora. The pelB gene is positioned betweentwo DraI restrictions sites; isolation of the gene is achieved bydigestion of plasmid pSH2111 with DraI and identification of a 1.9kilobase fragment on an agarose gel. The isolated fragment was thenligated into plasmid pUC8 which had been digested with SmaI. Theresultant plasmid is pSS1004 (see FIG. 41).

The plasmid pSS1004 was then treated with NdeI T₄ DNA polymerase andHind III, and the resultant fragment ligated into plasmid pIT2, whichhad been digested with NcoI, T₄ DNA polymerase and HindIII, to produceplasmid pSS1038 (FIG. 41). The plasmid pIT2 contains both the Salmonellatyphimurium araBAD promoter and the aid_(c) gene (Johnson et al.--Gene34: 137-145, 1985). The plasmid was then used to transform E. colistrain 706. The expression of the pelB gene on plasmid pSS1038 was thusexpected to be under the control of the araBAD promoter and turned on bythe presence of arabinose in the growth medium.

EXCRETION AND PURIFICATION OF PLB

E. coli cell 706 (F⁻, pro, thr, leu, argH, his, lac, phoSt, rpsL,lky-207) carrying plasmid pSS1038 was used to characterize theproduction and excretion of PLb. The E. coli cells were grown in TYE(1.5% tryptone, 1% yeast extract and 0.5% NaCl), incubated at 37° C., atlog phase of growth (around O.D.₅₄₀ =0.6), 1% of arabinose was added tothe growth medium to turn on the araBAD promoter and start producingPLb. After four hours of induction O.D.₅₄ was about 2.5), the culturebroth was centrifuged and PLb was directly purified from this E. colicell culture medium. The culture fluid was concentrated and desalted byAmicon (membrane YM2) and then the protein was purified by passingthrough a CM-52 column at pH 7.4 and then eluted with 0.2M NaCl. The PLpurified from these simple steps has greater than 95% purity, as judgedby electrophoresis on SDS gels followed by staining with Coomassie blue.

CONSTRUCTION OF THE SECRETION VECTOR

The pSS1004 plasmid was used as the source of the signal sequence forpelB. The sequence was isolated from pSS1004 digestion with restrictionenzyme HaeIII, ligation with SstI DNA linker, and then digestion withEcoRI. The EcoRI-SstI DNA fragment, which contains the 5'-end non-codingregion and the leader peptide was then ligated into a pBR322 plasmiddigested with SspI and EcoRI. The plasmid so produced containing-thesignal peptide, is pING173. FIG. 36 describes both the DNA sequence forthe signal peptide, and the procedure for preparing the pING173 plasmid.This plasmid is used to construct additional derivatives, as describedbelow.

PRODUCTION AND SECRETION OF THAUMATIN BACKGROUND

Thaumatin is a protein sweetener originally isolated from the plantThaumatococcus danielli. Thaumatin contains 207 amino acids and is2,000-5,000 fold sweeter than sucrose. It has eight disulfide bonds andthe tertiary structure of thaumatin is essential for its biologicalfunction.

CONSTRUCTION OF A PLASMID CARRYING A PECTATE LYASE B SIGNAL SEQUENCE ANDTHE SYNTHESIZED PLANT THAUMATIN GENE

The DNA sequence which codes for PLb signal peptide from plasmidpING173, described above was cloned in front of the thaumatin gene fromplasmid pING174 to secrete thaumatin in an E. coli host system. Theresulting plasmid, pING177-1, was used to express and secrete thaumatinin E. coli. It has the araBAD promoter and part of the arab gene fusedto 50 bp of the 5'-non-coding region of the PLb leader peptide. Toprepare this plasmid, the SstI and EcoRI fragment of plasmid pING173 wascloned into the plasmid pING174, which contains the thaumatin gene.pING174 was digested with BamHI and PstI, and the leader sequence frompING173 ligated into the restriction sites, to produce pING176. Thelatter plasmid was digested with NdeI and XhoI, and the resultingfragment, containing the pectate lyase leader sequence adjacent to thethaumatin gene, was cloned into the SalI, XhoI sites on the plasmidpING61. The resulting plasmid contained the gene coding for the PLbleader sequence and the thaumatin gene, under the control of the araBADpromoter and was referred to as pING177-1. The detailed constructionscheme is shown in FIG. 42).

PRODUCTION AND CHARACTERIZATION OF THAUMATIN FROM E. COLI RECOMBINANTSTRAIN

The E. coli strain 706 harboring plasmid pING177-1 was grown in oneliter of TYE broth. At O.D.=0.35, the cells were induced with 1% w/varabinose for approximately 12 hours. The culture was then harvested andthe periplasmic space protein was characterized by SDS-PAGE, Westernanalysis and the RIA assay for properly folded thaumatin. Both SDS-PAGEand Western analysis indicated that thaumatin could be synthesized by E.coli cells. RIA assay also indicated that the secreted thaumatin in theperiplasmic space of the E. coli was properly folded (Table 16). Thepre-thaumatin signal peptide was also used to secrete thaumatin in E.coli. Plasmid pING177-3, which contains the pre-thaumatin signal peptidesequence and the thaumatin structural gene, was used to producethaumatin, the detailed construction scheme is shown in FIG. 43). Theconditions for cell growth and induction are the same as those describedpreviously. The results of the RIA assay indicated that the productionof properly-folded thaumatin directed by the pre-thaumatin signalpeptide is less efficient than directed by the PLb leader peptide (Table16).

                  TABLE 16                                                        ______________________________________                                        Secretion of Thaumatin in Escherichia coli                                    (E. Coli K-12; 706)                                                                     RIA cross reactive Thaumatin (ug/                                             gram wet wt. cells                                                  Plasmid Media   Soluble Cell Extract                                                                          Total Secreted                                ______________________________________                                        177-1   5.4     35.2            40.6                                          177-3   1.8     6.3             8.1                                           ______________________________________                                    

What is claimed is:
 1. A polynucleotide molecule encoding animmunoglobulin fragment, said immunoglobulin fragment comprising a firstDNA sequence encoding a first pectate lyase secretion signal sequenceoperably linked to a DNA sequence encoding an immunoglobulin Fd moleculeor a fragment thereof comprising the variable region and a second DNAsequence encoding a second pectate lyase secretion signal sequenceoperably linked to a DNA sequence encoding an immunoglobulin light chainor a fragment thereof comprising the variable region, wherein said firstand second DNA sequences are operably linked to a single prokaryoticpromoter so as to form a dicistronic transcription unit, wherein saidimmunoglobulin fragment is capable of binding to an antigen and saidimmunoglobulin fragment is produced and secreted by an E. coli host cellwhen said polynucleotide molecule is expressed in said host cell.
 2. Avector comprising the polynucleotide molecule of claim
 1. 3. An E. colihost cell transformed with the vector of claim
 2. 4. The polynucleotidemolecule of claim 1, wherein said immunoglobulin Fd molecule or saidimmunoglobulin light chain are selected such that the resultantimmunoglobulin fragment is chimeric.